Journal: Gene therapy

Article Title: The use of polyethylenimine-DNA to topically deliver hTERT to promote hair growth.

doi: 10.1038/gt.2011.62

Figure Lengend Snippet: Figure 1 Flow cytometric analysis and fluorescence detection of sorted cells double stained for integrin b1 and CD 71. The integrin b1CD71 (a) and integrin b1+CD71 (b) cells were collected by FACS and examined by flow cytometry, respectively. Integrin b1CD71 cells are shown in the lower left-hand quadrant of each contour plot, whereas integrin b1+CD71 cells are shown in the lower right-hand quadrant, with percentage of each sorted cell population. Photo images of integrin b1CD71 (c) and integrin b1+CD71 cells (d). Integrin b1 was obvious by green fluorescence and CD71 was obvious by red fluorescence. Nuclei were counterlabeled with 4,6-diamidino-2-phenylindole (DAPI) (scale bar¼10 mm). The color reproduction of this figure is available on the html full text version of the manuscript.

Article Snippet: Stem cells, which expressed integrin a6, were detected by staining with anti-integrin a6 mouse monoclonal antibody (sc-59920; Santa Cruz Biotechnology, Inc.) followed by anti-mouse secondary antibody conjugated with Alexa Fluor 647 (A21235; Invitrogen) and with anti-hTERT with anti-rabbit secondary antibody conjugated with fluorescein isothiocyanate.

Techniques: Staining, Cytometry

Journal: Gene therapy

Article Title: The use of polyethylenimine-DNA to topically deliver hTERT to promote hair growth.

doi: 10.1038/gt.2011.62

Figure Lengend Snippet: Figure 3 hTERT expression in DNA–PEI complex transfected b1+CD71 keratinocytes increased expression of cell proliferation marker. (a–d) Detection of hTERT expression was detected by immunofluorescent staining (Allophycocyanin, APC) in integrin b1+CD71 keratinocytes after (a) treatment of water (Neg), (b) naked pLC–hTERT plasmid (DNA), (c) PEI vector only (PEI) and (d) PEI–pLC–hTERT complex at N/P¼7 (N/P). Panels (e–h) showed that immunofluorescent staining of PCNA (Allophycocyanin, APC) after treatment of water (e), DNA (f), PEI (g) and hTERT–DNA–PEI complex (h). Sections stained with antibodies that were color-coded according to the fluorescent tags of the secondary antibodies. Western blot analyses of hTERT (i) and PCNA (j) expression in cells transfected with the different treatments indicated on the top (Neg, water; DNA, pLC–hTERT plasmid; P7, PEI at molar ratio¼7; P15, PEI at molar ratio¼15; N/P7, hTERT–DNA–PEI complex at N/P ratio¼7; N/P15, hTERT–DNA–PEI complex at N/P ratio¼15). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control for normalization of the total protein amounts. The color reproduction of this figure is available on the html full text version of the manuscript.

Article Snippet: Stem cells, which expressed integrin a6, were detected by staining with anti-integrin a6 mouse monoclonal antibody (sc-59920; Santa Cruz Biotechnology, Inc.) followed by anti-mouse secondary antibody conjugated with Alexa Fluor 647 (A21235; Invitrogen) and with anti-hTERT with anti-rabbit secondary antibody conjugated with fluorescein isothiocyanate.

Techniques: Expressing, Transfection, Marker, Staining, Plasmid Preparation, Western Blot, Control

Journal: Nature Communications

Article Title: Control cell migration by engineering integrin ligand assembly

doi: 10.1038/s41467-022-32686-2

Figure Lengend Snippet: a Schematic illustration of precise control of integrin ligand presentation on nanofilaments via peptide assembly. b TEM images of nanofilaments obtained via molecular self-assembly and co-assembly of FFFIKLLI (100 μM) and FFF at various ratios, and the estimated molecular packing structures. IKLLI motif is presented in blue and FFF motif is presented in pink. The scale bars represent 200 nm. Three independent experiments were performed. c Zoom-in SEM images (false color) of HuH-7 cell edge and apical membrane after 3-day incubations. FFFIKLLI was maintained at a concentration of 100 μM. The cell body is highlighted in pink, while the nanofilaments are highlighted in blue. The scale bars represent 300 nm.

Article Snippet: Integrin β1 shRNA plasmid (#sc-29375-SH), Integrin α3 shRNA plasmid (#sc-35684-SH), Integrin α6 shRNA plasmid (#sc-43129-SH), and control shRNA plasmid-A (#sc-108060) were purchased from Santa Cruz Biotechnology for knockdown of the target integrins.

Techniques: Control, Membrane, Concentration Assay

Journal: Nature Communications

Article Title: Control cell migration by engineering integrin ligand assembly

doi: 10.1038/s41467-022-32686-2

Figure Lengend Snippet: a Time-lapse series showing actin cytoskeleton (grey) and paxillin (green) in HuH-7 cells expressing mRuby-Lifeact-7 and mGFP-paxillin upon the treatment of FFFIKLLI (100 μM) for 12 hr. Scale bars represent 2 μm. Three independent experiments were performed. b F-actin phalloidin staining (magenta), integrin β1 (cyan) and paxillin (yellow) immunofluorescence in HuH-7 cell after 12 h treatment of FFFIKLLI (100 μM). Scale bar represents 2 μm. c Fluorescence intensity distribution profile of integrin β1, paxillin, and actin cytoskeleton along the yellow line on merged image of b . d 12 h time course Rac1 activity of HuH-7 cells with or without the treatment of FFFIKLLI (100 μM). Rac1 activity was measured by FRET. n = 6 cells (Ctrl) or 5 cells (FFFIKLLI). Symbols represent the mean FRET/CFP emission ratio ± s.d. e Representative FRET/CFP ratio images of HuH-7 cells expressing RaichuEV-Rac1 with or without the treatment of FFFIKLLI (100 μM) at the indicated time points and coded according to a pseudo-color scale, which ranges from yellow to purple with an increase in Rac1 activity. Scale bars represent 20 μm. Source numerical data are available in source data.

Article Snippet: Integrin β1 shRNA plasmid (#sc-29375-SH), Integrin α3 shRNA plasmid (#sc-35684-SH), Integrin α6 shRNA plasmid (#sc-43129-SH), and control shRNA plasmid-A (#sc-108060) were purchased from Santa Cruz Biotechnology for knockdown of the target integrins.

Techniques: Expressing, Staining, Immunofluorescence, Fluorescence, Activity Assay

Journal: The Journal of Cell Biology

Article Title: Myc regulates keratinocyte adhesion and differentiation via complex formation with Miz1

doi: 10.1083/jcb.200506057

Figure Lengend Snippet: Comparison of the effects of MycER and MycV394DER activation on expression of proteins involved in keratinocyte adhesion. In all experiments, 200 nM 4-OHT was added to all cultures 5 d before harvesting. (A and B) Flow cytometry of cells labeled with antibodies to α6 (A) or β1 (B) integrin subunits. Note that the analysis was performed on cell pools. The heterogeneity in α6 expression most likely results from somewhat varying levels of Myc expression in individual cells. (C) Immunoblots probed with antibodies to the indicated proteins. (D) ChIPs demonstrating binding of Myc but not of MycV394D to the start sites of the integrin β1 and α6 genes in vivo. Chromatin from human keratinocytes infected with the indicated viruses was precipitated with control or α-Myc antibodies. For integrins β1 and α6, primers surrounding the transcription start site were used and, for nucleolin, primers surrounding the Myc-bound E-box sequence were used for the analysis.

Article Snippet: All were obtained from Santa Cruz Biotechnology, Inc. Immunoprecipitated DNA was amplified by PCR using primers specific for the transcription start site of the galectin, procollagen Iα2, integrin α6, and integrin β4 promoters and for the E-boxes of pcna, nucleolin, prothymosin-α, and β-tubulin promoters.

Techniques: Comparison, Activation Assay, Expressing, Flow Cytometry, Labeling, Western Blot, Binding Assay, In Vivo, Infection, Control, Sequencing

Journal: The Journal of Cell Biology

Article Title: Myc regulates keratinocyte adhesion and differentiation via complex formation with Miz1

doi: 10.1083/jcb.200506057

Figure Lengend Snippet: Effect of constitutive β1 integrin expression on the epidermal phenotype of mice with activated Myc. Dorsal epidermis was either unwounded (A–T) or wounded and analyzed 7 d later, after wound closure (U–X). (A–H and U–X) Hematoxylin and eosin staining; (I–L) immunostaining for Ki67; (M–P) immunostaining for keratin 10 (green) with DAPI nuclear counterstain; (Q–T) immunostaining for involucrin (green) with DAPI nuclear counterstain. Mice were either K14β1 (B, F, J, N, R, and V) or K14MycER (C, G, K, O, S, and W) single or double (D, H, L, P, T, and X) transgenics or wild-type littermate controls (A, E, I, M, Q, and U). Pictures were taken using a confocal microscope with a 25× lens with a numerical aperture of 0.80. Bars, 100 μm.

Article Snippet: All were obtained from Santa Cruz Biotechnology, Inc. Immunoprecipitated DNA was amplified by PCR using primers specific for the transcription start site of the galectin, procollagen Iα2, integrin α6, and integrin β4 promoters and for the E-boxes of pcna, nucleolin, prothymosin-α, and β-tubulin promoters.

Techniques: Expressing, Staining, Immunostaining, Microscopy

Journal: Virology Journal

Article Title: HPV16-E2 protein modifies self-renewal and differentiation rate in progenitor cells of human immortalized keratinocytes

doi: 10.1186/s12985-017-0736-2

Figure Lengend Snippet: HaCaT cell line possesses a “progenitor” sub-population. a Flow cytometric analyses for α6-integrin and CD71 expression in HaCaT cell line exhibited three phenotypes: α6-integrin dim (R8), α6-integrin bri /CD71 bri (R7) and α6-integrin bri /CD71 dim (R6). Upper panels correspond to enrichment assays sorting and reseeding only cells from the α6-integrin bri /CD71 dim subpopulation. Lower panels correspond to enrichment assays sorting and reseeding cells from a mix of α6-integrin dim (R8) and α6-integrin bri /CD71 bri (R7) subpopulations. b Bars graph of two rounds enrichment of the α6-integrin bri /CD71 dim subpopulation in self-renewal assays. Bars represent the mean ± SD of three independent assays ( p < 0.05). c Bars graph of the clonogenic assays from the α6-integrin bri /CD71 dim subpopulation seeded after each round of enrichment. Bars represent the mean ± SD of three independent assays ( p < 0.05). d RT-PCR analyses for the expression of stem cell markers ( SOX2 , OCT4 and NANOG ) in α6-integrin bri /CD71 dim subpopulation, Non-α6-integrin bri /CD71 dim subpopulation and total population in HaCaT cell line. e RT-qPCR analyses for the expression of SOX2 , OCT4 and NANOG in total HaCaTwt cells and after sorting in α6-integrin bri /CD71 dim and Non-α6-integrin bri /CD71 dim subpopulations. Values are expressed as the difference in ΔΔ Ct and expression of a housekeeping gene (β-actin). Relative expression of three separated assays expressed as the mean ± SD, p < 0.05, is presented. All images shown are representative of at least three independent experiments

Article Snippet: Cells were pelleted by centrifugation and suspended at 1 × 10 6 /100 μl in ice-cold PBS containing 1% BSA and then processed for single or double staining with PE-Cy5 mouse anti-human CD71 and PE rat anti-human α6 integrin (BD Biosciences, NJ, USA) during 45 min at 4 °C, using the appropriate negative controls to establish the compensation settings on the FACS.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Virology Journal

Article Title: HPV16-E2 protein modifies self-renewal and differentiation rate in progenitor cells of human immortalized keratinocytes

doi: 10.1186/s12985-017-0736-2

Figure Lengend Snippet: HPV16-E2 expression modifies the α6-integrin-CD71 subpopulations profile in HaCaT cell line. a RT-PCR analysis of lentivirus transduced HaCaT cells 5 days post-infection. HPV16-E2 is expressed only in transduced cells HaCaT-HPV16-E2. b Western blot analysis showing the expression of HPV16-E2 protein only in the transduced HaCaT-HPV16-E2 cells. c Representative flow cytometry analysis for the α6-integrin-CD71 subpopulations profile in HaCaT-HPV16-E2 cells. R6 decreases almost 50% while R8 increases at least 5 times in these cells. All images shown are representative of at least three independent experiments

Article Snippet: Cells were pelleted by centrifugation and suspended at 1 × 10 6 /100 μl in ice-cold PBS containing 1% BSA and then processed for single or double staining with PE-Cy5 mouse anti-human CD71 and PE rat anti-human α6 integrin (BD Biosciences, NJ, USA) during 45 min at 4 °C, using the appropriate negative controls to establish the compensation settings on the FACS.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Western Blot, Flow Cytometry

Journal: Virology Journal

Article Title: HPV16-E2 protein modifies self-renewal and differentiation rate in progenitor cells of human immortalized keratinocytes

doi: 10.1186/s12985-017-0736-2

Figure Lengend Snippet: HPV16-E2 expression alters the level of stem cell markers in HaCaT cells. RT-qPCR analyses for expression of SOX2 , NANOG and OCT4 in HaCaTwt, HaCaT-Vac and HaCaT-HPV16-E2 total cell population and after sorting in α6-integrin bri /CD71 dim and Non-α6-integrin bri /CD71 dim subpopulations. Values are expressed as the difference in ΔΔ Ct compared to non-infected cells and expression of a housekeeping gene (β-actin). Relative expression of three separated assays expressed as the mean ± SD, p < 0.05, is presented

Article Snippet: Cells were pelleted by centrifugation and suspended at 1 × 10 6 /100 μl in ice-cold PBS containing 1% BSA and then processed for single or double staining with PE-Cy5 mouse anti-human CD71 and PE rat anti-human α6 integrin (BD Biosciences, NJ, USA) during 45 min at 4 °C, using the appropriate negative controls to establish the compensation settings on the FACS.

Techniques: Expressing, Quantitative RT-PCR, Infection

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Integrin expression profiles in HT-1080 and MV3 cells and RNAi-based integrin targeting in HT-1080 cells. (A and B) Surface expression pattern of integrin β and α chains on HT-1080 (A) and β chains on MV3 (B) cells determined by flow cytometry. Black line, isotype control. Values, mean fluorescence (minus isotype values). (C–E) Downregulation of β1 and β3 integrins in HT-1080 cells by shRNA. (C) Knockdown efficiency of β1 integrin in dual-color HT-1080 cells (Western blot), compared with nontransduced (NT) and empty vector (EV)–transduced cells. β-Tubulin, loading control. (D and E) Upregulation of β3 integrins after downregulation of β1 integrin (D) and efficient downregulation of both β1 and β3 integrins after β1/β3RNAi (E) determined by flow cytometry. Surface expression pattern of β1 and β3 integrins on β1RNAi cells or β1/β3RNAi cells (red lines) compared with cells transfected with empty vector (EV, blue lines). Black line, isotype control. Values, mean fluorescence (minus isotype values). Stability of β1/β3 downregulation was routinely verified, and no outlier behavior or drift of expression to other integrin β-chains was noted (data not shown). (F) Reduction of β1 integrin adhesion epitope detected by FITC-conjugated mAb 4B4 on vector control (EV; left) and β1RNAi cells (right) after epitope saturation with unconjugated mAb 4B4 (3 µg/ml; blue line showing residual epitopes) compared with unspecific IgG1 (red line; total epitopes). Black line, isotype control staining (Iso). Values indicate mean fluorescence intensities. (G) Diminished phosphoErk signal (MAPK signaling) after β1/β3 integrin targeting (day 7). Histograms show the mean pixel fluorescence (MF) intensity of pErk from control (HT-1080 wild type) and β1/β3 integrin targeted tumors (T, dotted lines, identified by H2B-EGFP label) compared with pErk signal in the surrounding stroma (S), which further contained hair follicles (HF) with strong endogenous pErk expression. Ratio of tumor- and stroma-derived pErk intensity is displayed as medians, 25th/75th percentiles (box), and 5th/95th percentiles (whiskers) from one sample determined from 10 independent regions of the corresponding stroma region after exclusion of hair follicles, with a ratio of 1.0 (red dashed line) when signal intensity of both regions was equal. Calibration bar, pixel intensity. Scale bars, 100 µm (overview); 10 µm (inset).

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: Expressing, Flow Cytometry, Fluorescence, shRNA, Western Blot, Plasmid Preparation, Transfection, Staining, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Compromised tumor integrity and persistence of the invasion niche by RNAi- and antibody-based targeting of β1/β3 integrins in HT-1080 tumors. (A) Experimental procedure for administration of anti-β1 integrin mAb 4B4 or IgG1 and sequential intravital microscopy of the tumor response to integrin interference. Fluo, epifluorescence overview microscopy. MPM, subcellular-resolved multiphoton microscopy. (B) Time course of tumor growth or regression in control tumors transduced with empty vectors (p-puro/p-neo), β1RNAi or β1/β3RNAi in the absence or presence of IgG1 or anti-β1 integrin mAb 4B4. White arrowheads, onset of collective invasion. Numbers (right column), percentage mean regression of the tumor core (day 13 compared with day 6) from three to four independent tumors. Scale bars, 1 mm. (C) Fractions of mitotic and dead cells (day 6) quantified based on nuclear morphology for different interference schemes displayed as medians, 25th/75th percentiles (box), and 5th/95th percentiles (whiskers) from 20 independent fields from three to four independent tumors. Per condition, 19–20 nuclei were analyzed for the core and ∼10 nuclei for the invasion zone. *, P = 0.01; ***, P < 0.0001; ns, not significant. Statistics, Mann–Whitney U test (Bonferroni-corrected threshold: P = 0.0125). (D) Tumor development in response to the indicated interference procedures. Data show the means ± SD from three to four independent tumors. *, P = 0.0286. Statistics, Mann–Whitney U test. (E) Preferential survival of invading collective strands after combined β1/β3 integrin targeting. Z-projections of the same tumor region. Black box in upper left corner (day 6) results from stitching of adjacent images without complete overlap. Insets, mitotic figures in collective strands. Arrowheads, detachment of cell groups and individualized cells. Scale bar, 250 µm. (F) Median residual volume of tumor core and collective invasion (CI) zones after β1/β3RNAi combined with mAb 4B4 (day 13 compared with day 6) from three independent tumors. *, P < 0.05. Statistics, paired t test.

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: Intravital Microscopy, Microscopy, Transduction, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Radiosensitization of HT-1080 tumors by β1/β3 integrin RNA interference combined with antibody-based β1 integrin targeting. (A) Protocol for administration of anti-β1 (4B4) or IgG1 combined with fractionated IR and sequential intravital imaging of the tumor response. Fluo, epifluorescence overview microscopy. MPM, subcellular-resolved multiphoton microscopy. (B) Topology and extent of the invasion zone in response to fractionated IR combined with single-integrin (β1) or dual β1/β3 integrin interference. Epifluorescence (left) and 3D reconstructed z-projections from regions marked by dashed boxes using multiphoton microscopy (right; day 13). White asterisks, apoptotic nuclei. Scale bars, 1 mm (left); 250 µm (right). (C) Time-dependent tumor volume. Data show the means ± SD from three to four independent tumors, with P values for comparing irradiated integrin-targeted tumors to irradiated control tumors. (*), P = 0.006; *, P = 0.004. Statistics, Mann–Whitney U test (Bonferroni-corrected threshold: P = 0.005). (D) Regression of tumor core and collective invasion (CI) zone after IR with or without integrin mono- or dual interference. Data show median residual areas, 25th/75th percentiles (box), and 5th/95th percentiles (whiskers) of day 13 normalized to day 6. Per condition, four tumors were analyzed. (*), P = 0.03; ns, not significant. Statistics, Mann–Whitney U test (Bonferroni-corrected threshold: P = 0.0125).

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: Imaging, Microscopy, Irradiation, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Dual-integrin targeting abrogates radioresistance in the collective invasion niche. (A) Protocol for administration of anti-β1 (4B4) and αV integrin (17E6) mAbs or IgG1 combined with fractionated IR and sequential intravital imaging of the tumor response. Fluo, epifluorescence overview microscopy. MPM, subcellular-resolved multiphoton microscopy. (B) Radiation response of tumor core and collective invasion zone after combined treatment with mAbs 4B4 and 17E6 compared with IgG1-treated control (day 13). Black box in upper right corner (HT-1080, IgG 1 ) results from stitching of adjacent images without complete overlap. Asterisks, areas of regression. Arrows, persisting invasion strands. Alexa Fluor 660–conjugated dextran-perfused blood vessels. Second harmonic generation (SHG) originates from muscle and collagen fibers. Scale bar, 250 µm. (C) Frequency of dead cells in core and collective invasion (CI) zone after antibody-based integrin targeting and/or IR (day 6). Data show the medians, 25th/75th percentiles (box), and 5th/95th percentiles (whiskers) of ∼20 nuclei per condition and tumor regions from four to five tumors, reflecting a total of 9–28 different microscopic fields. *, P = 0.01; **, P = 0.001; ***, P < 0.0001; ns, not significant. Statistics, Mann–Whitney U test (Bonferroni-corrected threshold: P = 0.0125). (D) Extent of tumor regression in core and collective invasion zone of irradiated tumors combined with or without integrin targeting. Data show median residual areas, 25th/75th percentiles (box), and 5th/95th percentiles (whiskers) of day 13 normalized to day 6 from four independent tumors. *, P = 0.03. Statistics, Mann–Whitney U test.

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: Imaging, Microscopy, MANN-WHITNEY, Irradiation

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Radiosensitization of sarcoma and melanoma tumors by antibody-based integrin interference and procedures and outcome of long-term therapy response. (A–D) Tumor morphology and quantification of radiosensitization assessed by intravital microscopy. (A and B) Time-dependent growth or regression of HT-1080 or MV3 lesions in response to the indicated treatment conditions. n.a., not analyzed due to humane endpoint after day 13 (tumor >2 cm 3 ). Images of untreated HT-1080 and MV3 tumors are also shown in Fig. S1 A. Asterisks, regression tumor core. Arrowheads, tumor remnants. Scale bars, 1 mm. (C) Time-dependent tumor volume during and after treatment with IgG1 or mAb 4B4 + 17E6 with or without IR. Data show the means ± SD from three to four (HT-1080) or three to five (MV3) independent lesions. *, P = 0.0286 (comparison IgG1/IR control with 4B4/17E6 and IR [day 15]). Statistics, Mann–Whitney U test. (D) Mitotic frequencies in nonirradiated and irradiated tumor core and collective invasion (CI) zone. Data show the medians, 25th/75th percentiles (box), and 5th/95th percentiles (whiskers) of 10–20 nuclei per tumor region and condition from 7 to 23 independent fields from four (HT-1080) or three to five (MV3) independent tumors. ***, P < 0.0001. Statistics, Mann–Whitney U test. (E) Long-term follow-up (day 26) after treatment with 4B4 and 17E6 and IR, revealing minimal residual disease. Dotted gray line, position of former tumor. Box, position of lower panel. Zoom shows surviving cells without mitotic activity (arrowheads) and cytoplasm-free, condensed nuclei of disintegrated cells (asterisks). Scale bars, 250 µm. (F) Example tumor undergoing complete regression after therapy monitored longitudinally by whole-body fluorescence imaging. (G) IR dose escalation study for intradermal HT-1080 and MV3 tumors (window-free dermis). Left panels, overall survival after IR using the indicated doses (6–10 mice per group). Right panel, cure rate measured as percentage of tumors that did not relapse after IR. Black dashed line, IR dose with 30% cure rate (5 × 2 Gy for HT-1080 tumors, 5 × 3 Gy for MV3 tumors). (H) Dual-color detection of lung and lymph node metastases. Microscopic whole-organ screen (not depicted) was followed by analysis of cryosections (depicted). Scale bars, 100 µm (overview); 10 µm (inset). (I) Identification of minimal residual disease at the endpoint. The dorsal skin was screened from the deep fascia for presence or absence of fluorescent tumor remnants (left panel). In case of doubt, subregions were additionally sectioned for analysis by anti-EGFP immunohistochemistry (IHC; right panel). Images show typical tumor-negative outcome. Scale bar, 100 µm. (J) Examples of minimal residual lesions present at the tumor implantation site at the endpoint (day 180). Tumor remnants with strand-like pattern of green-fluorescent tumor nuclei (H2B-EGFP) followed by tissue sectioning and validation by anti-EGFP IHC. Dashed line, approximate position of tissue cross section. Arrowheads, intact H2B-EGFP–positive tumor nuclei. Right panel, validation of EGFP-positive tumor remnants and positive anti-EGFP IHC side by side. Scale bars, 100 µm (overview); 10 µm (details).

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: Intravital Microscopy, MANN-WHITNEY, Irradiation, Activity Assay, Fluorescence, Imaging, Immunohistochemistry, Tumor Implantation

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Dual-targeted but not individual anti-integrin therapy to enhance radiation response, tumor eradication, and long-term survival. (A) Treatment schemes for HT-1080 and MV3 tumors. Tumor cells were injected at day 0, resulting in an intradermally growing tumor located along the dorsal midline (dashed line). Example image, intradermal HT-1080 lesion. Time points of IR and antibody administration are indicated. (B) Tumor lesion (T) after implantation in imaging window–free mouse. Intradermal localization was confirmed by high-frequency ultrasound. (C) Collective invasion (CI) pattern in intradermal tumors in imaging window–free dermis (maximum-intensity projections). Number of multicellular strands per tumor was counted from 50-µm-thick tumor sections from nine (HT-1080) and seven (MV3) tumors. Scale bar, 100 µm. (D) Tumor-free overall survival of mice after application of treatment, including fractionated IR without and with individual and dual-targeted integrin inhibition with antibodies 4B4 and/or 17E6, compared with IR combined with isotypic control antibody (representing IR alone without integrin targeting). Mice were sacrificed after 180 d or earlier, upon humane endpoint criteria (tumor size of 2 cm 3 , ulceration, weight loss, or poor overall condition due to internal metastasis). See for details on mouse numbers (8–12 mice per group), metastasis formation, and tumor remnants. Gray-shaded area, therapy phase. *, P = 0.01; **, P = 0.0003; ***, P < 0.0001; ns, not significant. Statistics, log-rank survival analysis (Bonferroni-corrected thresholds: P = 0.01 [HT-1080] and P = 0.008 [MV3]).

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: Injection, Imaging, Inhibition

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Survival rates and tumor outcome for long-term follow-up experiments on integrin-targeted and IR therapy in HT-1080 sarcoma xenografts

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Collective cancer invasion forms an integrin-dependent radioresistant niche

doi: 10.1084/jem.20181184

Figure Lengend Snippet: Survival rates and tumor outcome for long-term follow-up experiments on integrin-targeted and IR therapy in MV3 melanoma xenografts

Article Snippet: Suspended cells were obtained by digestion with collagenase I (1,000 U/ml, 30 min, 37°C; Sigma-Aldrich), pelleted, and stained for 30 min on ice with the following monoclonal antibodies or isotypic control antibody: mouse anti-α1 integrin (clone TS2/7, 10 µg/ml; Abcam); mouse anti-α2 (CD49b, clone AK-7, 5 µg/ml), mouse anti-α3 integrin (CD49c, clone C3II.1, 0.25 µg/ml; both BD Biosciences); mouse anti-α5 integrin (clone SAM-1, 400 µg/ml; Millipore); rat anti-α6 integrin (CD49f, clone GoH3, 2.5 µg/ml; BD Biosciences); mouse anti-αV (CD51, clone AMF7, 50 mg/ml; Beckman Coulter); mouse anti-β1 (CD29, clone 4B4, 10 µg/ml), mouse anti-β2 (CD18, clone 7E4, 10 µg/ml), mouse anti-β3 integrin (CD61, clone SZ21, 15 µg/ml; all Beckman Coulter); rat anti-β4 (CD104, clone 439-9B, 0.625 µg/ml; BD Biosciences); mouse anti-β5 integrin (clone EM09902, 2 µg/ml; Absolute Antibody); mouse anti-β6 (clone 437211, 0.5 µg/ml; R&D Systems); rat anti-β7 integrin (clone FIB504, 0.3125 µg/ml; BD Biosciences); mouse anti-β8 integrin (clone 416922, 10 µg/ml; R&D Systems); and isotypic mouse IgG1κ (clone MOPC-21) and IgG2bκ (clone 27-35), rat IgG2aκ (clone R35-95), and IgG2bκ (clone A95-1; all BD Biosciences).

Techniques: