Journal: Cancers

Article Title: Fibroblasts Promote Resistance to KRAS Silencing in Colorectal Cancer Cells

doi: 10.3390/cancers16142595

Figure Lengend Snippet: List of anti-human antibodies used for flow cytometry.

Article Snippet: CD49f , APC , GoH3 , 130-100-147 , Miltenyi Biotec.

Techniques: Cytometry

Journal:

Article Title: Induction of ?3-Integrin Gene Expression by Sustained Activation of the Ras-Regulated Raf-MEK-Extracellular Signal-Regulated Kinase Signaling Pathway

doi: 10.1128/MCB.21.9.3192-3205.2001

Figure Lengend Snippet: Expression of β3- and α6-integrins in human cancer cell lines. CFPAC pancreatic cancer cells (A to D) or WM793 melanoma cells (E to H) were treated with either UO126, SB203,580, or DMSO as a solvent control for 48 h as indicated and then stained for the cell surface expression of β3- or α6-integrins by flow cytometry as described in Materials and Methods.

Article Snippet: Phycoerythrin (PE) or biotin-coupled anti-integrin antisera used for murine cell staining were purchased from Pharmingen: anti-β1-biotin (CD29, Ha2/5), anti-β3-PE (CD61, 2C9.G2), anti-β4 (CD104, 346-11A), anti-αv-biotin (CD51, H9.2B8), anti-α1 (CD49a, Ha31/8), anti-α2 (CD49b, HMa2), anti-α4-PE (CD49d, R1-2), anti-α5-PE (CD49e, 5H10-27), and anti-α6-PE (CD49f, GoH3).

Techniques: Expressing, Staining, Flow Cytometry

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:

Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Spatial Engineering of Mammary Epithelial Cell Cultures with 3D Bioprinting Reveals Growth Control by Branch Point Proximity

doi: 10.1007/s10911-024-09557-1

Figure Lengend Snippet: Extrusion 3D bioprinting of mammary epithelial cells inside an ECM leads to formation of polarized 3D cell cultures of a defined shape. A-B Immunofluorescence imaging of the BM protein laminin α5 (LAMA5, green) and nuclei (blue) in clonal spheroid cultures of MCF10A cells A , and 3D-bioprinted cultures of MCF10A and MCF10DCIS.com cells B on day 14. Magnified images are shown in the ROIs. The MCF10A spheroids embedded in an ECM with elevated BME content A produced results comparable to the ECM used in B . C-D Immunofluorescence imaging of the basal epithelial integrin α6 (ITGA6, green) and nuclei (blue) in clonal spheroid cultures of MCF10A cells C , and 3D-bioprinted cultures of MCF10A and MCF10DCIS.com cells D on day 14. Magnified images are shown in the ROIs. The MCF10A spheroids embedded in an ECM with elevated BME content C produced results comparable to the ECM used in D . E Immunofluorescence imaging of E-cadherin (green), vimentin (magenta) and nuclei (blue) in 3D-bioprinted cultures of MCF10A and MCF10DCIS.com cells on day 10. Magnified images are shown in the ROIs. F Immunofluorescence imaging of nuclei (DAPI) in the 3D-bioprinted cultures of MCF10A and MCF10DCIS.com cells on day 14. Some of the apoptotic cells with fragmented nuclei are indicated with yellow arrowheads. Scale bars A, C: 100 μm, ROI 25 μm; B, D, E: 500 μm, ROI 50 μm; F: 50 μm. Images represent the central plane of the cell cultures

Article Snippet: The gels were incubated with primary antibodies against integrin α6 (NKI-GoH3, MCA699GA, Bio-Rad, 1:800), laminin α5 (4C7, ab17107-1001, Abcam, 1:50), E-cadherin (24E10, 3195 S, Cell Signaling, 1:200), vimentin (V9, 347 M-1, Sigma, 1:1000) and Ki67 (GR3375556-1, ab15580, Abcam, 1:250) in the blocking solution for 3 days at 4 °C on a shaker.

Techniques: Immunofluorescence, Imaging, Produced