Journal: Nature Communications

Article Title: Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

doi: 10.1038/s41467-021-21858-1

Figure Lengend Snippet: a Dot plots representing uptake of Alexa 647-labeled iRGD-AgNPs or control AgNPs by mCherry-labeled CAF spheroids. The bar diagram shows the proportion of CAFs that internalized the AgNPs. n = 4 independent experiments. Two-tailed unpaired t test; p = 0.0008. b Dot plots representing iRGD-AgNP uptake by mCherry-labeled CAF spheroids in the presence of anti-NRP-1, αvβ3, or αvβ5 antibodies or control IgG. The bar diagram shows the proportion of CAFs that internalized the AgNPs normalized against IgG. n = 4 (Rabbit IgG and NRP-1), n = 5 (Mouse IgG, αvβ3, and αvβ5) independent experiments. One-way ANOVA; p = 0.4784 (NRP-1 vs. αvβ3), p < 0.0001 (NRP-1 vs. αvβ5 and αvβ3 vs. αvβ5). c Representative confocal images from three independent experiments of GFP-positive hPCF1424 CAFs (green) treated with Alexa 647-labeled iRGD-AgNPs (red) in the presence of mouse IgG (left panels) or an anti-αvβ5 blocking antibody (right panels). The cells were etched to remove the AgNPs bound to the surface and highlight the internalized particles. Pre-etch and post-etch images are shown. Scale bars 100 µm. d Bar diagram showing the % expression of αvβ5 or NRP-1 in hPCF1424 CAFs transiently transfected with non-specific siRNA (NS), two different pools of siRNAs against integrin β5 (ITGb5-1 and -2), or NRP-1 siRNA (siNRP-1), as measured by median fluorescence intensity (left panel). Representative data of three biological replicates. e Bar diagram showing flow cytometric analysis of iRGD-AgNP uptake by the siRNA-treated CAFs in ( d ). The results are shown as the proportion of CAFs that internalized the AgNPs. n = 3 (NRP-1), n = 5 (ITGb5-1 and -2) independent experiments. One-way ANOVA; p < 0.0001 (NS vs. siITGb5-1 and NS vs. siITGb5-2), p = 0.9997 (NS vs. siNRP-1). All error bars, SEM. *** p < 0.001; **** p < 0.0001. Source data provided in Source Data file.

Article Snippet: The sections were treated with a rabbit anti-αvβ5 polyclonal antibody (1:200) (Cat. no. bs-1356R, BIOSS, Woburn, MA) or a rabbit polyclonal anti-cleaved caspase 3 antibody (1:250) (Cat. no. 9579S, Cell Signaling) at 4 °C overnight and incubated with ImmPRESS (IHC-P, rabbit, MP-7401) secondary antibody reagent (Vector, Burlingame, CA).

Techniques: Labeling, Two Tailed Test, Blocking Assay, Expressing, Transfection, Fluorescence

Journal: Nature Communications

Article Title: Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

doi: 10.1038/s41467-021-21858-1

Figure Lengend Snippet: a Flow cytometry analysis showing entry of iRGD-AgNP or control AgNP into spheroids made of PC3 tumor cells alone or PC3 cells mixed with mCherry-labeled hPCF1424 CAFs (co-cult). Note that iRGD-AgNPs entered PC3 cells more efficiently in the presence of CAFs. b Quantified results of ( a ). Proportion of cells that internalized the particles are shown. n = 5 independent experiments. Two-tailed unpaired t test; p = 0.00015 (PC3 alone vs co-cult), p = 0.0177 (CAF alone vs. co-cult). c Flow cytometry analysis showing the expression of αvβ5 and αvβ3 integrins and NRP-1 in PC3 and CAF spheroids cultured alone (gray bars) or co-cultured with each other (black bars). n = 6 independent experiments. Two-tailed unpaired Student’s t test; p = 0.000325 (αvβ5: PC3 alone vs. co-cult), p = 0.0247 (αvβ5: CAF alone vs. co-cult), p = 0.0158 (αvβ3: PC3 alone vs. co-cult), p = 0.0132 (αvβ3: CAF alone vs. co-cult), p = 0.947 (NRP-1: PC3 alone vs. co-cult), p = 0.055 (CAF alone vs. co-cult). All error bars, SEM. * p < 0.05; *** p < 0.001. Source data provided in Source Data file.

Article Snippet: The sections were treated with a rabbit anti-αvβ5 polyclonal antibody (1:200) (Cat. no. bs-1356R, BIOSS, Woburn, MA) or a rabbit polyclonal anti-cleaved caspase 3 antibody (1:250) (Cat. no. 9579S, Cell Signaling) at 4 °C overnight and incubated with ImmPRESS (IHC-P, rabbit, MP-7401) secondary antibody reagent (Vector, Burlingame, CA).

Techniques: Flow Cytometry, Labeling, Two Tailed Test, Expressing, Cell Culture

Journal: Nature Communications

Article Title: Tumor-penetrating therapy for β5 integrin-rich pancreas cancer

doi: 10.1038/s41467-021-21858-1

Figure Lengend Snippet: a Expression of αvβ5 integrin in PC3 cells after 2 days of incubation in normal media (NM) or CM prepared from cultured hPCF1424 CAFs ( n = 9), mPCFAA0779 CAFs ( n = 7), or MIA PaCa-2 human PDAC cells ( n = 4) performed in independent experiments. Fold over NM is shown. One-way ANOVA; p < 0.0001 (NM vs. hPCF1424 CM), p = 0.5085 (NM vs. mPCFAA0779 CM), p = 0.9886 (NM vs. MIA PaCa-2). b Dot plots representing iRGD-AgNP or control AgNP uptake in PC3 cells cultured in NM or CM from hPCF1424 CAFs. c The bar diagrams show the proportion of PC3 (left) or LM-PmC (right) cells that internalized iRGD-AgNPs. The cells were incubated in NM or CM from hPCF1424 CAFs for 2 days prior to study. n = 6 (PC3), n = 3 (LM-P) independent experiments. Two-tailed unpaired Student’s t test; p = 0.0001 (PC3 NM vs. CAF CM), p = 0.0127 (LM-P NM vs. CAF CM). d Expression of actin, αv, and β5 integrin mRNAs normalized against cyclophilin A analyzed by qPCR in PC3 cells incubated with NM or CM from hPCF1424 CAFs. n = 3 independent experiments. Two-tailed unpaired Student’s t test; p = 0.00016 (αv), p < 0.0001 (β5), p = 0.923 (actin). e , f Expression of αvβ5 integrin ( e ) or NRP-1 ( f ) in PC3 cells cultured in normal media (NM) or CM from hPCF1424 CAFs in the presence or absence of exogenous TGF-β, a TGF-β-specific inhibitor LY2157299, or vehicle alone. Mean fluorescence intensity (MFI) assessed by flow cytometry is shown. n = 4 independent experiments. Two-tailed unpaired Student’s t test; p = 0.0008 ( e: NM vs. TGF-β), p = 0.0009 ( e: TGF-β vs. TGF-β + LY2157299), p = 0.7082 ( e: CAF CM vs. CAF CM + DMSO), p = 0.0177 ( e: CAF CM + DMSO vs. CAF CM + LY2157299). No significant differences in ( f ). All error bars, SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Source data provided in Source Data file.

Article Snippet: The sections were treated with a rabbit anti-αvβ5 polyclonal antibody (1:200) (Cat. no. bs-1356R, BIOSS, Woburn, MA) or a rabbit polyclonal anti-cleaved caspase 3 antibody (1:250) (Cat. no. 9579S, Cell Signaling) at 4 °C overnight and incubated with ImmPRESS (IHC-P, rabbit, MP-7401) secondary antibody reagent (Vector, Burlingame, CA).

Techniques: Expressing, Incubation, Cell Culture, Two Tailed Test, Fluorescence, Flow Cytometry

Journal: British Journal of Cancer

Article Title: Binding of TGF-β1 latency-associated peptide (LAP) to αvβ6 integrin modulates behaviour of squamous carcinoma cells

doi: 10.1038/sj.bjc.6600545

Figure Lengend Snippet: Cell adhesion to LAP is αvβ6-dependent. Chromium [ 51 Cr]- labelled cells (1.5×10 4 ) were added to LAP-coated 96-well plates containing an irrelevant control antibody (W632 anti-MHC class 1) or test antibodies against αv (L230), αvβ5 (P1F6), αvβ6 (10D5), β1 (P4C10) and α5β1 (P1D6). Background binding to BSA has been subtracted from the results. Figures show representative experiments performed in quadruplicate. Error bars represent standard deviation. ( A ) VB6 cells show increased adherence LAP. β6 transfected cells (VB6), control cells (C1) and αv-negative cells (H357) were plated onto varying concentrations of LAP. VB6 cells showed significantly increased adhesion compared to C1 cells (which express low levels of endogenous αvβ6). H357 cells did not adhere. ( B ) Adhesion to LAP is αvβ6-dependent. VB6 and C1 adhesion to LAP was inhibited by anti-αvβ6 antibody or anti-αv antibody. Antibodies against αvβ5, α5β1 or β1 produced no effect. These data suggest that adhesion of VB6 and C1 cells is modulated solely through αvβ6.

Article Snippet: Anti-αvβ5 (P1F6) was obtained from Life Technologies, Paisley, UK.

Techniques: Binding Assay, Standard Deviation, Transfection, Produced

Journal: British Journal of Cancer

Article Title: Binding of TGF-β1 latency-associated peptide (LAP) to αvβ6 integrin modulates behaviour of squamous carcinoma cells

doi: 10.1038/sj.bjc.6600545

Figure Lengend Snippet: Cell migration towards LAP is αvβ6-dependent. Cells were allowed to migrate towards LAP in haptotactic migration assays. To assess integrin specificity of migration, integrin-blocking antibodies against αv (L230), αvβ5 (P1F6), αvβ6 (10D5), or a control antibody (W632) were added to VB6, C1 and H357 cells prior to plating into wells. Following 8 h incubation, the cells in the lower chamber (including those attached to the undersurface of the membrane) were trypsinised and counted on a Casy 1 counter (Sharfe System GmbH, Germany). Results for the cell lines are expressed relative to VB6 migration following incubation with an irrelevant control antibody (=100). Figures show representative experiments performed in quadruplicate. Error bars represent standard deviation. ( A ) VB6 cells show increased migration towards LAP. Comparison of migration of VB6, C1 and H357 cells towards LAP. Migration is higher in the αvβ6 expressing VB6 cells. αv-negative H357 cells did not migrate significantly. ( B ) Migration towards LAP is αvβ6-dependent. Migration of VB6 and C1 cells was inhibited completely by antibody inhibition of αvβ6 or αv. Antibodies inhibiting αvβ5 produced no effect. These data suggest that migration of VB6 and C1 cells is modulated solely through αvβ6.

Article Snippet: Anti-αvβ5 (P1F6) was obtained from Life Technologies, Paisley, UK.

Techniques: Migration, Blocking Assay, Incubation, Standard Deviation, Expressing, Inhibition, Produced

Journal: ACS Nano

Article Title: Dynamic Surfaces for the Study of Mesenchymal Stem Cell Growth through Adhesion Regulation

doi: 10.1021/acsnano.6b01765

Figure Lengend Snippet: Integrin, BMP2 receptor, and cytoskeletal tethering changes in MSCs on dynamic surfaces. (A) Integrin β 1 and BMPR1a staining in MSCs cultured on FMOC-RGD (low) at t 0 ( t 0 is after 48 h of culture immediately before addition of elastase) and then after 24 and 48 h post-elastase treatment (cleaved RGD) or in FMOC-RGD surfaces. β 1 was observed to be found in punctate adhesions, with little BMPR1a colocalization noted (48 h inset). On the cleaved RGD (high) surfaces, BMPR1a was seen with adhesion morphology but in different areas to the regions of β 1 localization (48 h outset). (B) Integrin β 5 and BMPR1a staining in MSCs on FMOC-RGD surfaces at t 0 (immediately before addition of elastase) and then at 24 and 48 h post-elastase (cleaved RGD) or in FMOC-RGD surfaces. On the FMOC-RGD surfaces, little β 5 expression and no BMPR1a colocalization were observed. However, on the cleaved surfaces, strong β 5 /BMPR1a colocalization was noted by 24 h (outset images). (C) Actin/ezrin colocalization could be seen at t 0 and on FMOC-RGD. However, for cleaved RGD samples, ezrin appeared to colocalize with cortical actin at the cell periphery (arrows). SiRNA knock-down of ezrin resulted in an increase in pRUNX 2 5 days post-switch. For pRNUX2, in-cell western analysis, n = 3, results are mean ± SD, stats by ANOVA and Dunn’s post-hoc test where * P < 0.05.

Article Snippet: After fixation, samples were incubated with a primary antibody cocktail consisting of rabbit polyclonal anti-BMPR1A (Thermo Scientific; 1:50), mouse monoclonal anti-integrin β1 (Thermo Scientific; 1:50), and mouse anti-integrin αvβ5 (R&D system, 1:50) in 1% of BSA/PBS.

Techniques: Staining, Cell Culture, Expressing, In-Cell ELISA