Journal: Oncogene

Article Title: EPHB6 augments both development and drug sensitivity of triple-negative breast cancer tumours

doi: 10.1038/s41388-018-0228-x

Figure Lengend Snippet: EPHB6 inhibits scattering of TNBC cells. a EPHB6-deficient TNBC cells, MDA-MB-231, were stably transfected with expression vectors encoding EPHB6 (MDA-B6), myc-tagged EPHB6 (MDA-B6-M), or mock-transfected with an empty vector (MDA-pc3). EPHB6 expression was analysed by Western blotting; tubulin and ERK2 represent loading controls. b MDA-pc3, MDA-B6 and MDA-B6-M cells were grown in individual colonies on glass coverslips in 24-well plates. Colonies were fixed and stained with phalloidin (red) and DAPI (blue). Representative colonies were imaged using an Olympus FV1000 confocal microscope with a 10× objective lens. Scale bar, 100 µm, inserted using ImageJ. c Individual colonies of MDA-pc3, MDA-B6 and MDA-B6-M cells were fixed and stained with crystal violet in 6-well plates. Scattered and compact colonies were counted in six wells per cell line, using an inverted microscope. The graph represents the percentage of scattered colonies in each cell line. d TNBC cells, BT-20, were transduced with EPHB6-targeting shRNAs (shB6-1 or shB6-2, individually), as indicated. Transduction with non-silencing shRNA (BT20-NS) was used as a control. EPHB6 expression was analysed as in a , and quantitated by densitometry. EPHB6 quantifications were normalised on matching tubulin controls and presented in arbitrary units (AU). e Formation of scattered and compact colonies by BT20-NS, BT20-shB6-1 and BT20-shB6-2 cells was analysed as in c . f Representative confocal microscopy images of BT20 cells with intrinsic EPHB6 expression (BT20) and of MDA-B6-M cells (MDA-B6-M) both co-stained with anti-EPHB6 (red) and anti-ZO-1 (green). Stainings with matching non-specific IgGs are shown as specificity controls. Images were captured using an LSM 700 Zeiss confocal microscope with 40× oil objective lens. Scale bar, 20 μm, was inserted using ZEN 2012 software. Red and green signal intensities were set using matching non-specific IgG controls as thresholds. Panels were generated using PowerPoint and Adobe Illustrator CS6 software. Data are shown as means ± SD. Experiments were performed at least three times; * P < 0.05; Student’s t -test

Article Snippet: To transduce cells with other shRNA constructs (Santa Cruz: shOCT4-1, sc36123-SH; shErk2-1, sc35335-SH; Sigma: shEPHB6-2 (shB6-2), TRCN0000010677; shOCT4-2 TRCN0000004882; shErk2-2, TRCN0000010040) and previously described GFP or RFP constructs [ ], procedures were performed as described for the Luciferase assay.

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Western Blot, Staining, Microscopy, Inverted Microscopy, Transduction, shRNA, Confocal Microscopy, Software, Generated

Journal: Oncogene

Article Title: EPHB6 augments both development and drug sensitivity of triple-negative breast cancer tumours

doi: 10.1038/s41388-018-0228-x

Figure Lengend Snippet: EPHB6 increases proliferation of tumoursphere-producing cells. a Cells were seeded into Ultra-Low Attachment 24-well plates (4 × 10 3 cells per well) to form tumourspheres for 7 days. Tumourspheres were dissociated and analysed by flow cytometry for Ki-67 in duplicates. Gating was based on matching isotype control. The graph represents analysis of three independent experiments and shows percentage of Ki-67-positive cells in each cell line. b Cells were labelled with 10 µM BrdU for 48 h in monolayers using BrdU kit (R&D Systems). Initial levels of BrdU incorporation were examined by flow cytometry. Remaining cells were seeded in the BrdU-free medium to proliferate in tumourspheres for 7 days, as in a . Tumourspheres were dissociated and BrdU loss was assessed by flow cytometry. The graph represents the analysis of three independent experiments and shows fold decrease in mean intensity of BrdU staining in each cell line relative to matching initial measurements. c To directly assess the rate of proliferation of tumoursphere-forming cells, MDA-pc3 or MDA-B6-M cells were seeded into 24-well Ultra-Low attachment plates (4 × 10 3 cells per well) and allowed to propagate in tumourspheres for 7 days. Representative tumourspheres were imaged. For each replicate, tumourspheres from 12 independent wells were pulled together, dissociated into a single-cell suspension, counted and the average number of cells per well was calculated. The graph represents the analysis of three independent experiments performed in duplicates. d The indicated cells were propagated in tumourspheres and analysed as in c . e TNBC cells, HCC70, were transduced with non-silencing shRNA (HCC70-NS) or EPHB6-targeting shRNA, shB6-1, and EPHB6 expression was analysed as in Fig. . f The indicated cells were propagated in tumourspheres and analysed as in c . g GFP- or RFP-expressing HCC70-NS cells (HCC70-NS-GFP or HCC70-NS-RFP) were mixed in equal numbers (1:1 ratio) with RFP- or GFP-expressing HCC70-shB6-1 cells (HCC70-shB6-1-RFP or HCC70-shB6-1-GFP), as indicated and allowed to proliferate in tumourspheres for 6 days. Samples of mixed cells were collected at day 0 and day 6, and analysed by flow cytometry and the FlowJo software in triplicates. In addition, RFP-expressing MDA-B6-M (MDA-B6-M-RFP) cells mixed in equal numbers (1:1 ratio) with GFP-expressing MDA-pc3 (MDA-pc3-GFP), cells were propagated and analysed as described above for HCC70. Each graph represents analysis of triplicates and shows the proportion of GFP- and RFP-expressing cells, as indicated for each combination, at seeding (Day 0) and after 6 days of propagation in tumourspheres. Solid lines in the graph indicate mean values. h EpCAM expression was analysed by flow cytometry. Data are shown as means ± SD. Experiments were performed at least three times; * P < 0.05; Student’s t -test or Mann–Whitney U -test. Scale bars, 1000 µM. For optimal presentation, individual tumoursphere images are shown at different brightness and contrast settings

Article Snippet: To transduce cells with other shRNA constructs (Santa Cruz: shOCT4-1, sc36123-SH; shErk2-1, sc35335-SH; Sigma: shEPHB6-2 (shB6-2), TRCN0000010677; shOCT4-2 TRCN0000004882; shErk2-2, TRCN0000010040) and previously described GFP or RFP constructs [ ], procedures were performed as described for the Luciferase assay.

Techniques: Flow Cytometry, BrdU Incorporation Assay, BrdU Staining, Transduction, shRNA, Expressing, Software, MANN-WHITNEY

Journal: Oncogene

Article Title: EPHB6 augments both development and drug sensitivity of triple-negative breast cancer tumours

doi: 10.1038/s41388-018-0228-x

Figure Lengend Snippet: EPHB6 relies on elevated OCT4 expression to enhance proliferation of tumoursphere cells. a Total cell lysates were prepared from the indicated cells using Lysis Buffer from the Proteome Profiler Stem Cell Array Kit (R&D) and OCT4 expression was assessed by Western blotting. b MDA-B6-M and BT-20 cells were stably transduced with non-silencing shRNA or OCT4-targeting shRNAs (shOct4-1 or shOct4-2, individually), as indicated. OCT4 expression was assessed by Western blotting with anti-OCT4 and quantitated as in Fig. 2d. c The indicated cells were seeded into 96-well Ultra-Low attachment plates (2 × 10 3 cells per well), allowed to propagate in tumourspheres for 7 days and analysed as in Fig. . Graphs represent analyses of three independent experiments. Data are shown as means ± SD. Experiments were performed at least three times; * P < 0.05; Mann–Whitney U -test. Scale bars, 1000 µM. For optimal presentation, individual tumoursphere images are shown at different brightness and contrast settings

Article Snippet: To transduce cells with other shRNA constructs (Santa Cruz: shOCT4-1, sc36123-SH; shErk2-1, sc35335-SH; Sigma: shEPHB6-2 (shB6-2), TRCN0000010677; shOCT4-2 TRCN0000004882; shErk2-2, TRCN0000010040) and previously described GFP or RFP constructs [ ], procedures were performed as described for the Luciferase assay.

Techniques: Expressing, Lysis, Western Blot, Stable Transfection, Transduction, shRNA, MANN-WHITNEY

Journal: Oncogene

Article Title: EPHB6 augments both development and drug sensitivity of triple-negative breast cancer tumours

doi: 10.1038/s41388-018-0228-x

Figure Lengend Snippet: EPHB6 accelerates tumour growth and supports tumour initiation. a , b MDA-pc3 and MDA-B6-M cells were inoculated into the mammary fat pad region of 4–6-week-old female athymic nude mice ( n = 7 per group; 1.5 × 10 6 cells per mouse) ( a ) or NOD-SCID mice ( n = 7 per group; 1.5 × 10 5 cells per mouse) ( b ), and tumour growth was monitored, as indicated. Tumour volume was calculated by the equation: A /2× B 2 , where A was long and B was short diameter of the tumour. c MDA-pc3 and MDA-B6-M were injected into NOD-SCID mice as in b ( n = 4 per group). Tumours were processed for staining with anti-CD34 or haematoxylin and eosin (H&E) staining. For immunohistochemistry, tumours were fixed in 10% formalin, paraffin embedded and 4-µm-thick sections were affixed on slides. Dewaxing and antigen retrieval were performed using the Dako PT Link system (Dako Canada Inc., Mississauga, ON, Canada). Staining was performed on Dako Autostainer Link using anti-CD34 and the Dako FLEX DAB+ Detection Kit. In each section, 12, 3, 6 and 9 o’clock fields were imaged with a Nikon Eclipse E400 microscope (Nikon Instruments Inc., Melville, NY, USA) at 100× magnification and blood vessel density was quantified using the ImageJ software. The graph summarises two independent experiments. Scale bar, 100 µm. d Decreasing numbers of MDA-pc3 or MDA-B6-M were injected into NOD-SCID mice and tumour formation was monitored for 60 days. The graph summarises two independent experiments. At least five animals per condition were used in each experiment. e HCC70-NS and HCC70-shB6-1 cells were injected into NOD-SCID mice ( n = 5 per group; 1.5 × 10 6 cells per mouse) and tumour growth was monitored. Upon experiment termination, mice were photographed; scale bar, 10 mm. Tumours were photographed and weighed. f Tumour formation by decreasing numbers of HCC70-NS or HCC70-shB6-1 in NOD-SCID mice was analysed as in d . The graph summarises data from two independent experiments. At least five animals per condition were used in each experiment. Data are shown as means ± SD. Animals were randomly assigned to all experimental groups. Experiments were performed at least two times. TIC frequencies were estimated using the Extreme Limiting Dilution Analysis ; * P < 0.05; Student’s t -test or Mann–Whitney U -test; n.s. statistically not significant

Article Snippet: To transduce cells with other shRNA constructs (Santa Cruz: shOCT4-1, sc36123-SH; shErk2-1, sc35335-SH; Sigma: shEPHB6-2 (shB6-2), TRCN0000010677; shOCT4-2 TRCN0000004882; shErk2-2, TRCN0000010040) and previously described GFP or RFP constructs [ ], procedures were performed as described for the Luciferase assay.

Techniques: Injection, Staining, Immunohistochemistry, Microscopy, Software, MANN-WHITNEY

Journal: Oncogene

Article Title: EPHB6 augments both development and drug sensitivity of triple-negative breast cancer tumours

doi: 10.1038/s41388-018-0228-x

Figure Lengend Snippet: EPHB6 relies on the ERK pathway for executing its responses in tumourspheres. a Phosphorylation status of the indicated proteins was examined by Western blotting with phospho-specific antibodies. Blotting or re-blotting of the same membranes with anti-tubulin was used as a loading control. For anti-p-JNK analysis, equal aliquots of matching cell lysates were loaded twice on the same gel and blotted with either anti-phospho-JNK or anti-tubulin, since p-JNK and tubulin signals overlapped. b Phosphorylation of ERK kinases was assessed by Western blotting. c The indicated cells were lysed and immunoprecipitations were performed with anti-GRB2 or a matching amount of non-specific control antibody. Immunoprecipitates were resolved by non-reducing SDS PAGE and EPHB6 presence was analysed by Western blotting. Presence of GRB2 in immunoprecipitates or input lysates was monitored by Western blotting. Removed irrelevant lanes are indicated by dashed lines. d The indicated cells were serum starved for 24 h and analysed for Ras activation using the G-LISA Ras Activation Assay Biochem Kit (Cytoskeleton Inc). Ras activation is presented in optical density units (OD). Graphs represent analyses of three independent experiments. e Cells were analysed by Western blotting for activating c-Raf phosphorylation at Ser338. f Cells were treated with U0126 (10 μM) or DMSO at 37 °C for 2 h and phosphorylation status of ERK kinases was assessed by Western blotting. g MDA-B6-M and BT-20 cells were cultured for 72 h with U0126 (10 μM) or a matching volume of DMSO, and OCT4 expression was assessed by Western blotting. h Proliferation of tumoursphere cells in the presence of U0126 (10 μM) was examined as in Fig. 5c. i MDA-B6-M cells were transduced with non-silencing shRNA (MDA-B6-M-NS) or ERK2-targeting shRNA, shErk2-1. ERK2 expression was assessed as in Fig. . The effect of ERK2 silencing on cell proliferation in tumourspheres was analysed as in Fig. . j OCT4 expression was monitored by Western blotting. Data are shown as means ± SD. Experiments were performed at least three times; * P < 0.05; Student’s t -test or Mann–Whitney U -test. Scale bars, 1000 µM. For optimal presentation, individual tumoursphere images are shown at different brightness and contrast settings

Article Snippet: To transduce cells with other shRNA constructs (Santa Cruz: shOCT4-1, sc36123-SH; shErk2-1, sc35335-SH; Sigma: shEPHB6-2 (shB6-2), TRCN0000010677; shOCT4-2 TRCN0000004882; shErk2-2, TRCN0000010040) and previously described GFP or RFP constructs [ ], procedures were performed as described for the Luciferase assay.

Techniques: Western Blot, SDS Page, Activation Assay, Cell Culture, Expressing, Transduction, shRNA, MANN-WHITNEY

Journal: Oncogene

Article Title: EPHB6 augments both development and drug sensitivity of triple-negative breast cancer tumours

doi: 10.1038/s41388-018-0228-x

Figure Lengend Snippet: EPHB6 reduces tumour drug resistance. a MDA-pc3 and MDA-B6-M were injected into the mammary fat pad of NOD-SCID mice (1.5 × 10 6 cells per mouse). Mice with tumours were treated weekly with i.v. injections of doxorubicin (2 mg/kg) or saline ( n = 5 per group). Day 0 indicates the day of the initial treatment. The reduction in tumour growth in doxorubicin-treated mice is presented as a percentage relative to matching saline-treated controls. Upon experiment termination, tumours were photographed and weighed. Only four animals per group were included in these final measurements, due to earlier tumour-unrelated lethality. The middle graph represents tumour weights in doxorubicin-treated mice as a percentage relative to matching saline-treated controls. Average tumour weights for each group of experimental animals are shown in the lower graph. b HCC70-NS and HCC70-shB6-1 were injected into NOD-SCID mice and effectiveness of doxorubicin treatment was assessed as in a . Data are shown as means ± SD. Animals were randomly assigned to all experimental groups. Experiments were performed at least two times; * P < 0.05; Student’s t -test or Mann–Whitney U -test; n.s. statistically not significant

Article Snippet: To transduce cells with other shRNA constructs (Santa Cruz: shOCT4-1, sc36123-SH; shErk2-1, sc35335-SH; Sigma: shEPHB6-2 (shB6-2), TRCN0000010677; shOCT4-2 TRCN0000004882; shErk2-2, TRCN0000010040) and previously described GFP or RFP constructs [ ], procedures were performed as described for the Luciferase assay.

Techniques: Injection, MANN-WHITNEY

Journal: Oncogene

Article Title: EPHB6 augments both development and drug sensitivity of triple-negative breast cancer tumours

doi: 10.1038/s41388-018-0228-x

Figure Lengend Snippet: EPHB6 controls the behaviour of TNBC patient-derived xenograft. a – d Cells isolated from a TNBC patient-derived xenograft, HCI-010, were transduced with non-silencing shRNA (HCI-010-NS) or EPHB6-targeting shRNA (shB6-1) and analysed by Western blotting for EPHB6 ( a ), vimentin ( b ) and OCT4 ( c ) expression; proliferation of these cells in tumourspheres was analysed as in Fig. , scale bar, 1000 µM ( d ). e Representative images of HCI-010-NS and HCI-010-shB6-1 tumourspheres shown at a higher magnification; scale bar, 400 µM. f HCI-010-NS and HCI-010-shB6-1 cells were analysed by flow cytometry to determine the proportion of aldehyde dehydrogenase 1 (ALDH1)-positive cells using the ALDEFLUOR assay kit (Stemcell Technologies). The assay was performed according to the manufacturer’s instructions. Representative flow cytometry profiles including the negative controls (cells treated with DEAB, a specific ALDH1 inhibitor) are shown. The graph represents the analysis of triplicates and shows the proportion of ALDH1-positive cells in each cell line after subtracting the background values obtained from the matching negative controls. Solid lines in the graph indicate mean values. The data represent one of two independent experiments. g ERK phosphorylation was monitored by Western blotting. h Proliferation of HCI-010 cells in tumourspheres in the presence of U0126 (10 μM) was analysed as in Fig. ; scale bar, 1000 µM. Effect of U0126 on ERK phosphorylation was confirmed by Western blotting. i Proliferation of HCI-010 cells in tumourspheres in the presence of PD0325901 (100 nM) was analysed as in Fig. ; scale bar, 1000 µM. Effect of PD0325901 on ERK phosphorylation was confirmed by Western blotting. j HCI-010 cells were transduced with ERK2-targeting shRNA (shErk2-1). ERK2 silencing was confirmed as in Fig. . Proliferation of cells in tumourspheres was analysed as in Fig. ; scale bar, 1000 µM. Data are shown as means ± SD. Experiments were performed at least three times until otherwise indicated; * P < 0.05; Student’s t -test or Mann–Whitney U -test. For optimal presentation, individual tumoursphere images are shown at different brightness and contrast settings

Article Snippet: To transduce cells with other shRNA constructs (Santa Cruz: shOCT4-1, sc36123-SH; shErk2-1, sc35335-SH; Sigma: shEPHB6-2 (shB6-2), TRCN0000010677; shOCT4-2 TRCN0000004882; shErk2-2, TRCN0000010040) and previously described GFP or RFP constructs [ ], procedures were performed as described for the Luciferase assay.

Techniques: Derivative Assay, Isolation, Transduction, shRNA, Western Blot, Expressing, Flow Cytometry, MANN-WHITNEY

Journal: Oncogene

Article Title: EPHB6 augments both development and drug sensitivity of triple-negative breast cancer tumours

doi: 10.1038/s41388-018-0228-x

Figure Lengend Snippet: EPHB6 enhances growth of TNBC tumours and improves their drug sensitivity. a – c HCI-010-NS and HCI-010-shB6-1 cells were injected into the mammary fat pad region of 4–6-week-old NOD-SCID mice (1 × 10 6 cells per mouse). Mice with tumours were treated with doxorubicin or saline ( n = 6 per condition) as in Fig. . Growth rates of saline-treated HCI-010-NS and HCI-010-shB6-1 tumours were compared ( a ). Doxorubicin-induced reductions in growth of HCI-010-NS and HCI-010-shB6-1 tumours are presented as a percentage relative to matching saline-treated controls ( b ). Upon termination of the experiment at day 46 following initial cell injections, the excised tumours were photographed and weighed ( c ). Tumour images from one of two independent experiments are presented. The left graph in c represents tumour weights in doxorubicin-treated mice as a percentage relative to matching saline-treated controls. Average tumour weights for each group of experimental animals are shown in the right graph. The graphs represent analysis of two independent experiments. d Kaplan–Meier curve showing the probability of recurrence-free survival in 581 patients with basal breast cancer. The lower tertile of EPHB6 expression level was used as a cutoff. The 95% confidence interval for the hazard ratio (HR) is shown in parentheses. Probabilities for patients with EPHB6 levels in their tumours above the lower tertile are shown in red, while the probability for those with EPHB6 levels below the lower tertile are shown in black. Data are shown as means ± SD. Animals were randomly assigned to all experimental groups. Experiments were performed at least two times; * P < 0.05; Student’s t -test or Mann–Whitney U -test; n.s. statistically not significant

Article Snippet: To transduce cells with other shRNA constructs (Santa Cruz: shOCT4-1, sc36123-SH; shErk2-1, sc35335-SH; Sigma: shEPHB6-2 (shB6-2), TRCN0000010677; shOCT4-2 TRCN0000004882; shErk2-2, TRCN0000010040) and previously described GFP or RFP constructs [ ], procedures were performed as described for the Luciferase assay.

Techniques: Injection, Expressing, MANN-WHITNEY