Tumour growth inhibition and anti-metastatic activity of a mutated furin-resistant Semaphorin 3E isoform

Secreted Semaphorin 3E (Sema3E) promotes cancer cell invasiveness and metastatic spreading. The pro-metastatic activity of Sema3E is due to its proteolytic fragment p61, capable of transactivating the oncogenic tyrosine kinase ErbB2 that associates with the Sema3E receptor PlexinD1 in cancer cells. Here, we show that a mutated, uncleavable variant of Sema3E (Uncl-Sema3E) binds to PlexinD1 like p61-Sema3E, but does not promote the association of PlexinD1 with ErbB2 nor activates the ensuing signalling cascade leading to metastatic spreading. Furthermore, Uncl-Sema3E competes with endogenous p61-Sema3E produced by tumour cells, thereby hampering their metastatic ability. Uncl-Sema3E also acts independently as a potent anti-angiogenic factor. It activates a PlexinD1-mediated signalling cascade in endothelial cells that leads to the inhibition of adhesion to extracellular matrix, directional migration and cell survival. The putative therapeutic potential of Uncl-Sema3E was validated in multiple orthotopic or spontaneous tumour models in vivo, where either local or systemic delivery of Uncl-Sema3E-reduced angiogenesis, growth and metastasis, even in the case of tumours refractory to treatment with a soluble vascular endothelial growth factor trap. In summary, we conclude that Uncl-Sema3E is a novel inhibitor of tumour angiogenesis and growth that concomitantly hampers metastatic spreading.


Knock down of gene expression by RNA-interference. The expression of
(fused with a polyHis-Myc-epitope tag at C'-term) were subcloned into the lentiviral transfer plasmid pRRLsin.cPPT.hCMV.GFP.Wpre and gene transfer was perfomed as previously described (Follenzi et al 2002) This method ensured stable gene transfer with very high efficiency, without need to select individual cell clones. Control cells 65°C to inactivate endogenous phosphatases, and incubated with NBT-BCIP (nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl-phosphate) AP substrate (Promega).
Binding competition assay was performed on overexpressing plexinD1 COS cells treated with p61-Sema3E fused to SeAP plus increasing amount of 7nM,15nM,60nM). Cells were then washed three times, fixed with acetoneformaldehyde, and incubated at 65°C for 15 min to inactivate endogenous phosphatases. Cell-bound p61-Sema3E was quantified by measuring SeAP activity on p-nitrophenylphosphate hydrolysis at 37°C in a reaction buffer containing 1M Tris-HCl pH 9.5, 1% bovine serum albumin, 1 mM MgCl 2 . Absorbance values were measured at a wavelength of 405 nm and corrected for background absorbance.
Purified Uncl-Sema3E production. Affinity-purified Uncl-Sema3E, applied for treatment in all in vitro and in vivo experiments (except those based on gene transfer, systemic naked DNA hydroporation or lentiviral vector delivery), was obtained as follows. We subcloned murine Sema3E cDNA mutated in the furin cleavage site into a lentiviral expression vector (NSPI) including a human immunoglobulin Fc tag fused at the C'-terminal. HEK293 cells transfected to express the protein were grown in DMEM high glucose (4.5 g/l) medium containing 10% FCS. The conditioned medium collected from the culture was filtered to remove debris and Uncl-Sema3E-Fc protein was affinity purified by using protein G sepharose beads (Sigma). After sample loading, the column was extensively washed, including in the presence of 300mM NaCl to remove non-specific binding proteins. The purified protein was finally eluted in presence of 100mM Glycine pH 3, subsequently neutralized, snap frozen and stored at -80 • C. For systemic in vivo delivery experiments, tumor-bearing mice were randomized (four days after transplant) and received either 30mg/kg of purified Uncl-Sema3E or the same amount of bovine albumin (as control), injected intraperitoneally twice a week.
Immunopurified proteins or equal amounts of total cellular lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blocked in phosphatebuffered saline, 0.1% Tween, 10 % BSA. The membrane was then incubated with primary antibodies, followed by the appropriate peroxidase-conjugated secondary antibody (Bio-Rad). Final detection was done by enhanced chemiluminescence (ECL, Amersham Biosciences).
Cell migration assays. Cell motility was assayed using Transwell® chamber inserts (Costar, Cambridge, MA) with a porous polycarbonate membrane (8 μM pore size) as described previously (Barberis et al 2004). To allow haptotactic migration, the lower side of the filter was coated with 10 μg/ml fibronectin. Cells were harvested from culture dishes by treatment with 1mM EDTA and resuspended in 0.2% BSAcontaining medium. Approximately 1x10 5 cells were added in the upper chamber, and allowed to migrate through the filter towards the lower chamber (including regulatory molecules, when indicated) for 6-8 hours, in a cell culture incubator. For experiments with primary pericytes we included 3.5x10 4 cells in the upper chamber and the assay was performed in 0.1% FBS-containing medium. Eventually, the cells adherent to the upper side of the filter were mechanically removed, while those migrated to the lower side were fixed with 11% glutaraldehyde and stained with crystal violet. The dye was then solubilized in 10% acetic acid to measure absorbance at 595nm in a microplate reader (OD values are shown in the graphs as arbitrary units).

Immunofluorescence analysis of endothelial cells.
HUVECs were cultured overnight on glass coverslips, previously coated with 10µg/µl fibronectin (Sigma). After the indicated treatments, cells were fixed with 4% paraformaldehyde (PAF) in PBS for 20 min on ice. Cells were then permeabilized for 5 min on ice with 0.2% Triton X-100 in PBS, washed and incubated with blocking buffer (PBS supplemented with 2% goat serum, Vector Laboratories Inc.) for 30 min at room temperature. Primary antibodies were incubated with cells for 30 min; secondary antibodies were conjugated with AlexaFluor dyes (Molecular Probes). F-actin was stained with Phalloidin-FITC (Sigma) and nuclei with DAPI (Roche). Immunofluorescence images were captured using a Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems), by maintaining constant laser power, gain and offset settings. Digital images were evaluated by computer assisted analysis employing Image-ProPlus 6.2 software. For the quantification of active-β1-Integrin, we quantified mean fluorescence intensity in the respective channel in at least four independent broad microscopic fields (over several adjacent focal levels) per each experimental condition by means of the Leica Confocal Software Histogram Quantification Tool.

Cytofluorimetric analysis of Uncl-Sema3E induced endothelial cell apoptosis.
HUVECs grown for 24 hours in gelatin-coated dishes were treated with 20 nM Uncl-

Time-lapse microscopy analysis of living cells.
For the dynamic analysis of endothelial cell collapse induced by Uncl-Sema3E, time-lapse video-microscopy analysis was performed using a Leica AF6000LX workstation. HUVEC were plated into glass-bottomed dishes (WillCo-dish; Willcowells) and placed onto a sample stage within an incubator chamber set at 37 °C in an atmosphere of 5% CO2 and 20% humidity. Phase contrast images were captured with a 10× objective every 2 minutes, keeping lamp intensity at minimum to avoid phototoxicity. Movies were generated by employing the LAS AF Leica Application Suite software (Leica).

Cell proliferation analysis.
Tumor cells were seeded in multiple 96-well dishes (Costar) at an initial density of 2×10 3 cells per well. After 16 hours, culture medium was changed and the cells were grown in presence of 0.5% FBS for the following days. Every 24 hours, one multiwell dish was fixed with 11% glutheraldehyde, stained with crystal violet, and the absorbance was read using a standard colorimetric system at 562nm.

Histological analysis.
OCT-embedded frozen samples were cut in 10 µm thick sections and probed with primary antibodies (according to standard methods). Subsequent detection was done by HRP-or fluorescent-conjugated secondary antibodies (Alexa 488 or 546, Molecular Probes). Immunofluorescence images were captured using a Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems), by maintaining constant laser power, gain and offset settings. In certain experiments, a Leica DM IRBM microscope was also used. Digital images were evaluated by computer assisted analysis employing Image-ProPlus 6.2 or Metamorph software. Unless otherwise indicated, quantification was done by analyzing at least 5 sections and 10 fields per tumor (considering 6 tumors per experimental condition). The extent of pericyte coverage was quantified by drawing a region of interest (ROI) close to each CD31 + blood vessel, and calculating the percentage of co-staining in the two channels detecting CD31 and αSMA markers.
The statistical significance of results was verified by calculating p values with Student's t-test.

Assessment of tumor vessel perfusion, vessel permeability and tissue hypoxia.
Vessel perfusion was revealed upon intravenous injection of 0.05 mg/mouse FITClabeled lectin (Lycopersicon esculentum; Vector Laboratories) 10 minutes before sacrifice and tumor excision. Vessel permeability was analyzed by intravenous coinjection of 0.25 mg Texas Red-conjugated Dextran 70 kD (Molecular Probes) and 0.05 mg FITC-labeled lectin (as above) in anesthetized mice; ten minutes later, the animals were perfused with saline and 2% PAF; the tumors were then harvested and frozen in optimum cutting temperature (OCT) compound. Tumor hypoxia was revealed upon injection of 60 mg/kg pimonidazole hydrochloride into tumor-bearing mice 2 hours before sacrifice. To detect the formation of pimonidazole adducts, tumor sections were immunostained with hypoxyprobe-1-Mab1 (Chemicon) following the manufacturer's instructions.
Experiments in RIP-Tag2 mouse model. Generation of RIP-Tag2 mice as a model of pancreatic -cell carcinogenesis has been previously reported (Hanahan 1985).
RipTag2 mice were maintained in the C57Bl/6J background (The Jackson Laboratory). We analyzed at least eight tumors per each condition, in multiple experiments. Alzet osmotic minipumps (2002 model, Charles River Laboratories) were used to accomplish local delivery of Uncl-Sema3E in the pancreas of experimental mice. Osmotic minipumps were incubated in physiological saline for 2 h at 37 C° before implantation, and then filled with saline or a solution of purified Uncl-Sema3E calculated to deliver to the mice a dose of 1 mg/Kg/day over a period of 14 days (between 12-14 week of age). Osmotic minipumps were connected to a silastic tube (25 mm length, 1.65 mm external diameter, 0.7 mm inner diameter) and implanted subcutaneously in the right side of abdomen, under anesthesia with isofluorane 1.5%. The attached tube was passed into the peritoneal cavity and sutured to the abdominal wall to reach directly the pancreas. The proximity of tube tip to the pancreas was re-assessed at the time of sacrifice. Sunitinib L-malate (Axon Medchem BV) was administered to mice daily by oral gavage, at a dose of 40 mg/Kg. Total Tumor growth and lung metastatsis were evaluated as described in main manuscript.

Blood tests.
Blood tests in mice were performed according to standard analytical methods. Briefly, blood samples were collected via intra-cardiac puncture, in presence of Na Citrate to avoid blood clotting, and analyzed using photometric method (Modular Roche diagnostic). A. The conditioned medium (CM) of 4T1 tumor cells either control-EV, overexpressing Uncl-Sema3E, or knocked down for Sema3E expression by RNAi, was harvested and concentrated 1000-times, and eventually analyzed by immunoblotting with an anti-Sema3E antibody to reveal endogenous p61 levels.

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The CM of Sema3E-depleted cells provided a control of band specificity. B. The conditioned medium of MDA-MB435 cells expressing p61-Sema3E (myc-tagged), or coexpressing p61 and Uncl-Sema3E (Fc-tagged), were subjected to pull-down experiments using protein-G beads in order to capture Uncl-Sema3E. Immunoblotting analysis revealed that, although p61 was abundantly present in the conditioned media of co-expressing cells (see "input" lanes), it was not specifically found in association with Uncl-Sema3E ("pull-down lanes"). The first two lanes on the left were loaded with purified proteins to provide reference bands. C. Affinity purified Uncl-Sema3E (Fc-tagged, lane A) and p61-Sema3E (lane B) were allowed to interact in cell culture medium (lane C), and the mix was subjected to to pull-down experiments using protein-G beads in order to capture Uncl-Sema3E (lanes A', B', C'). Coomassie blue protein staining revealed that p61 did not specifically associate with Uncl- Sema3E

D C
A-B: 4T1 and A549 tumor cells overexpressing Uncl-Sema3E (and respective controls transduced with empty vector, EV) were analyzed in vitro to reveal potential growth rate changes in culture medium containing 0.5% FBS. C-E: Uncl-Sema3E overexpressing 4T1 cells (or EV cells treated with purified Uncl-Sema3E for 24 hours) did not show significant changes in the expression of typical markers of epithelial-mesenchymal transition (EMT) compared to controls, as revealed by western blotting (C). The same was true in A549 cells, as shown by expression analysis with western blotting (D) or Q-PCR (E); notably, cells treated with 5 ng/ml TGF-β (for 24h) underwent major phenotypic changes, and were included as internal positive controls of EMT. Uncl-Sema3E downregulates active integrin-β1 in endothelial cells. HUVEC cells were treated (or not) with 7nM Uncl-Sema3E for 5' and then incubated with antibodies detecting integrin-β1 molecules in the active conformation on the cell surface (green channel); signal quantification is shown in the graph (see Methods for details); **p<0.002. After fixation, the cells were furthermore stained to reveal Paxillin (red), F-actin (blue) and the nuclei with DAPI (shown in white). Scale bars: 50 µm.

Control untreated
Pre-treated with anti-active-β1-Int. + Uncl-Sema3E Not pre-treated + Uncl-Sema3E   Histological analysis of pancreatic tumors from RT2 mice (14 wk) either untreated or treated with locally-delivered Uncl-Sema3E. A. The mitotic index was determined by immunostaining Ki67 + cells and counterstain all nuclei with DAPI. B. Vessel coverage with mural cells was assessed by double staining for α-SMA and CD31 markers. C. Endothelial cell apoptosis in tumor vessels was assessed by double staining for CD31 and activated-caspase3 markers. In addition, the fraction of act-casp3+ apoptotic cancer cells (vs. DAPI staining, not shown) was increased in Uncl-Sema3E treated tumors; ***p<0.0005. D. Confocal images of PlexinD1-expressing cells (green) in untreated tumors. Endothelial cells were labelled by Meca32 (red); DAPI was used to stain nuclei. Scale bars throughout the figure: 100μm.

Control Uncl-Sema3E Sunitinib
Uncl-Sema3E anti-angiogenic activity did not induce increased metastatic spreading. A. The presence of tumor cells in loco-regional lymph nodes (LN) of RT2 mice treated with Uncl-Sema3E (n=14) or Sunitinib (n=8) or mock untreated (n=12) was assessed by immunostaining for SV40 T-antigen (representative images are shown on the left; scale bars: 200µm). The graph on the right indicates the mean tumor burden present in LN (±SD); ***p<0.0001. Below the graph is indicated the overall incidence of tumor-infiltrated LN in the different conditions; the difference vs. controls is significant for the Sunitinib treated arm (by the chi-square test: p<0.05). B. Liver tissue sections of RT2 mice treated with Uncl-Sema3E or Sunitinib or mock untreated (as above) were immunostained for SV40 T-antigen to detect the presence of metastatic tumor cells. We occasionally found isolated micrometastatic foci in control RT2 mice (2 out of 12) and Uncl-Sema3E-treated mice (2 out of 14). In contrast, the anti-angiogenic activity of Sunitinib was associated with remarkably increased incidence of distant metastasis in the liver (6 out 8 analyzed mice; p<0.0005, by the chi-square test). Microscopic images display the few metastatic cases in control and Uncl-Sema3E treated mice, as well as representative images of the liver of Sunitinibtreated mice; scale bars: 200µm). *** 4T1 cells were transduced to stably express shRNAs targeting PlexinD1 (or unrelated control sequences), and the expression knock down was verified by Q-PCR (shown on top left). PlexinD1deficient and control cells were then transplanted into nude mice to establish orthotopic tumors and the mice were subjected to systemic treatment with purified Uncl-Sema3E-Fc or control (similarly to experiments shown in main Fig. 9). The graphs show the growth of primary tumors (A), the total tumor burden (B) and the number of lung metastasis (C) at the end of the experiment. Data are given as average ±SD of six mice for each experimental group; ***p<0.0005.