Journal: Cancers

Article Title: Breast Tumor Cell Invasion and Pro-Invasive Activity of Cancer-Associated Fibroblasts Co-Targeted by Novel Urokinase-Derived Decapeptides

doi: 10.3390/cancers12092404

Figure Lengend Snippet: Macroscopic view of whole lungs and hematoxylin and eosin (H&E)-stained lung sections from Pep 2-treated mice. ( A ) Representative images of lungs surgically removed from healthy animals or from animals intravenously injected with HT1080 cells: of these, 5 were untreated (vehicle), 5 were treated with Pep 2 either at 15 mg/kg or at 3.4 mg/kg. ( B ) H&E-stained of the corresponding representative lung sections with the neoplastic areas marked with T (4× magnification, Scale bar 100 µm. ( C ) 50 ng of total genomic DNA extracted from lung samples from each vehicle, or Pep 2-treated mice were used as template for semi-quantitative PCR reactions and compared to the healthy lungs (H). The amplification products are separated on 1% agarose gels. A standard curve was included in every run, generated by mixing 10 7 , 10 6 ,10 5 ,10 4 ,10 3 ,10 2 ,10 1 ,1 HT1080 human cells with 1 to 10 7 murine P19 cells (Standard 1 to 9). The uncropped image for agarose gel can be found in . ( D ) The densitometric analysis of PCR amplification products, accomplished by acquiring images and quantifying them through ImageJ software (NIH, Bethesda, MD, USA), was performed on three independent gels. The signal from the 1 × 10 7 HT1080 cells sample was considered as 100% and standards 2, 3, 4 and 5 were graded relative to that (** p < 0.005; *** p < 0.001, Student’s t -test).

Article Snippet: The HT1080 human fibrosarcoma cell line was also purchased from the ICLC Interlab Cell Line Collection.

Techniques: Staining, Injection, Real-time Polymerase Chain Reaction, Amplification, Generated, Agarose Gel Electrophoresis, Software

Journal: Cancers

Article Title: Breast Tumor Cell Invasion and Pro-Invasive Activity of Cancer-Associated Fibroblasts Co-Targeted by Novel Urokinase-Derived Decapeptides

doi: 10.3390/cancers12092404

Figure Lengend Snippet: Fibrosarcoma cell invasion in the presence of fibroblasts pre-exposed to Pep 1 or Pep 2 in 3D-Organotypic assays. ( A ) Time scale representation of the 3D-organotypic invasion assay. 5 × 10 4 TIF fibroblasts/sample were first embedded into a neutralized collagen I solution and incubated for 3 days. Then 1 × 10 5 HT1080-GFP cells were seeded on the matrices, grown for 2 days (T0) and let to invade for 3 or 6 days. Matrices were then processed as described in . ( B ) Representative images of matrices sections after DAPI staining, 20× magnification, Scale bar, 50 µm. Fibroblasts were processed as described in ( A ), in the absence of Pep 2 (NT). Duplicate samples were exposed to 100 nM Pep 2 during HT1080-GFP invasion (Pep 2 invasion) or to 100 nM Pep 1 or Pep 2 during matrix contraction (Pep 1 contraction, Pep 2 contraction) and then peptides were removed and the matrices washed twice with growth medium. During contraction or invasion peptides were added to matrices every day. ( C ) The number of invading cells was quantified by densitometric analysis of images shown in B, with ImageJ software, the number of HT1080-GFP invading cells on untreated matrices was normalized to T0 and the 3 days sample was considered as 100%. ( D ) 5 × 10 4 TIF fibroblasts were seeded in DMEM-10% FBS in 6 well plates for 24 h, serum-starved for 6 h and incubated for 24 h in presence or in the absence of 100 nM Pep 1 or Pep 2 peptides in DMEM-10%FBS. Peptides were then removed and, after two PBS 1× washes, cells were incubated in serum-free medium for 24 h. Conditioned media (CM) from TIFs exposed to Pep 1 or Pep 2 were collected and employed as a chemoattractants for H1080-GFP cells in Boyden chamber migration assays. In each sample, 2 × 10 4 HT1080-GFP cells were tested either toward DMEM-0.1% BSA (basal migration) or toward TIFs CM. Basal migration was taken as 100% and all values were calculated relative to that. * p < 0.05; ** p < 0.005; *** p < 0.001, Student’s t -test.

Article Snippet: The HT1080 human fibrosarcoma cell line was also purchased from the ICLC Interlab Cell Line Collection.

Techniques: Invasion Assay, Incubation, Staining, Software, Migration

Journal: Cancers

Article Title: Breast Tumor Cell Invasion and Pro-Invasive Activity of Cancer-Associated Fibroblasts Co-Targeted by Novel Urokinase-Derived Decapeptides

doi: 10.3390/cancers12092404

Figure Lengend Snippet: Pep 1 and Pep 2 peptides inhibit HT1080 invasion by binding to αv integrin subunit. ( A ) 2 × 10 4 HT1080 cells/sample or ( B ) 5 × 10 4 /sample TIF fibroblasts were pre-incubated with or without 100 nM Pep 1 or Pep 2 for 1 h at 37 °C and then assayed in Boyden chambers at 37 °C, either for FBS-dependent invasion on matrigel for 5 h ( A ) or for FBS-dependent migration for 3 h ( B ). Data are expressed as in the legend to D (*** p < 0.001, Student’s t -test). ( C ) 2 × 10 6 HEK-293 cells, or HEK-293-αv-38, TIF or HT1080 cells were pre-incubated for 30 min at 4 °C with an excess of unlabeled Pep 2 or scrambled peptides (500 nM) and exposed to 50 nM FITC-Pep 2 for 2 h at 4 °C. Cell surface-associated fluorescence, as percentage of the samples with no FITC-Pep 2, is shown. Data represent a mean of three independent experiments performed in duplicate (*** p < 0.001, Student’s t -test). ( D ) HT1080 cells were transiently transfected with si-control (si-CTRL) or si-RNA targeting αv integrin mRNA (si-αv). The efficiency of silencing was assessed by Western Blot using polyclonal anti-αv antibody or monoclonal anti-GAPDH as loading control and quantified by Image J (inset). The whole blot image can be found in . After 48 h, 2 × 10 6 cells/sample were analysed for FITC-Pep 2 specific binding, as described in panel ( C ). As indicated, HT1080 cells were pre-treated with anti-αv polyclonal (Ab), or monoclonal antibody (Mab) or anti-actin polyclonal or anti-GAPDH monoclonal antibodies or with Pep 2 or 200 nM vitronectin (VN) for 1 h at 37 °C and analyzed for FITC-Pep 2 specific binding. Data represent a mean of three independent experiments performed in duplicate (** p < 0.005; *** p < 0.001, Student’s t -test).

Article Snippet: The HT1080 human fibrosarcoma cell line was also purchased from the ICLC Interlab Cell Line Collection.

Techniques: Binding Assay, Incubation, Migration, Fluorescence, Transfection, Control, Western Blot, Bioprocessing

Journal: Cancers

Article Title: Breast Tumor Cell Invasion and Pro-Invasive Activity of Cancer-Associated Fibroblasts Co-Targeted by Novel Urokinase-Derived Decapeptides

doi: 10.3390/cancers12092404

Figure Lengend Snippet: Decreased pro-invasive ability, matrix contraction capacity, and α-SMA protein levels in αv-silenced TIF fibroblasts. ( A ) Organotypic assay conducted with 5 × 10 4 TIF fibroblasts, transfected with siRNA CTRL or si-αv, combined with a collagen I solution. 1 × 10 5 HT1080-GFP cells were seeded on top of these matrices, incubated for 2 days (T0) and let to invade for 3 or 6 days. Matrices were processed as described in the legend to . Scale bar 50 µm. ( B ) The number of invading HT1080-GFP cells in ( A ) was quantified, normalized to T0 and reported as percentage of si-CTRL fibroblasts after 3 days, considered as 100%. ( C ) 5 × 10 4 TIF fibroblasts, transfected with si-CTRL or si-αv, were grown for 48 h in DMEM-10% FBS and serum-starved for 24 h. CM were collected and employed as chemoattractants for HT1080-GFP in Boyden chamber assays as described in the legend of D. ( D ) Contraction assay carried out with 5 × 10 4 TIF fibroblasts transfected with siRNA CTRL or siRNA to integrin αv and, after 24 h, mixed with the collagen I solution. The reduction of matrix diameter was monitored for 3 days. The area of matrices with TIF fibroblasts transfected with si-CTRL at day 2 was taken as 100%. ( E ) Western blot of total lysates from fibroblasts transfected with siRNA CTRL or siRNA to αv integrin were collected and subjected to quantitation of α-SMA and αv protein levels using GAPDH as a reference. The whole blot image can be found in . ( F ) 2 × 10 4 fibroblasts were seeded in DMEM-10% FBS on cover slips in 6 well plates for 24 h and silenced for integrin αv expression by RNA interference. After 72 h, cells were fixed and stained with DAPI or anti-α-SMA antibody by immunofluorescence (20× magnification and 63× in the inset). Scale bar 50 µm. The MFI following staining with anti-α-SMA antibody was quantified and reported as indicated in the legend to D. * p < 0.05; ** p < 0.005; *** p < 0.001, Student’s t -test.

Article Snippet: The HT1080 human fibrosarcoma cell line was also purchased from the ICLC Interlab Cell Line Collection.

Techniques: Transfection, Incubation, Contraction Assay, Western Blot, Quantitation Assay, Expressing, Staining, Immunofluorescence