Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: Overexpression of ROR1/YAP1 in breast cancer is associated with poor overall survival outcomes. (a) Graphical representation of cancer types in which ROR1 is most frequently altered in the TCGA dataset. (b) ROR1 and YAP1 mutation analysis of 960 breast cancer samples in the TCGA dataset. (c) Significant co-occurrences of ROR1 and YAP1 in the TCGA dataset. (d) Boxplot comparing ROR1 and YAP1 expression in ER + PR+/HER2-; ER + PR+/HER2+; ER-PR-/HER2+; subtypes samples in the TCGA dataset (one-way ANOVA). (e) Immunohistochemical detection of ROR1 and YAP1 in disease free, ER + PR+/HER2-; ER + PR+/HER2+; ER-PR-/HER2+; breast cancer subtypes from our own data cohort (one-way ANOVA). The boxes show the median ±1 quartile, with whiskers extending to the most extreme data point within 1.5 interquartile ranges from the box boundaries. (f) Survival analysis of ROR1 and YAP1 high and low expressing breast cancer patients based on expression intensity (Log-rank test; p < .0001, scale bar: 50 μM). (g) Bar graph showing the ROR1 and YAP1 expression intensity (%) in treatment naïve, sensitive, partially sensitive and resistant patient tumors (**p < .0, two-tailed Student's t -test).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Over Expression, Mutagenesis, Expressing, Immunohistochemical staining, Two Tailed Test

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: Treatment resistant BC cells overexpress ROR1 have a higher sphere forming efficiency: (a) Administration of therapy leads to the increase of ROR1 expression intensity in HER2 + treatment naïve and treatment exposed BC patient ( p = 3.021e-02. unpaired two-sided t -test). (b) Tumor cells were dissociated from treatment naïve, sensitive and resistant HER2 + BC patients and equal numbers of cells were cultured in the supplemented CSC medium and stained for ROR1 expression in the spheres. Matched HER2 + BC patient's tissues were stained for ROR1 expression by immunofluorescence (Scale bar: 50 μM). (c) FACS analysis of ROR1 + and ROR1 − population in treatment naïve, sensitive and resistant cells from primary HER2 + BC patient tumors. (d) Equal number of sorted ROR1 + and ROR1 − cells from primary BC patients ( n = 2) were cultured in the supplemented CSC medium and analyzed for sphere forming efficiency ( p < .0002, paired two-sided t -test). (e) Comparison of epithelial-to-mesenchymal transition related genes showing mRNA expression from treatment naïve, sensitive and resistant ROR1 + and ROR1 − primary HER2 + BC patients cell fractions (results are mean fold change +/− SE, n = 3). (f) Comparison of ROR1 mRNA expression in primary HER2 + treatment naïve, sensitive and resistant BC patient tumors (results are mean fold change +/− SE, n = 3). (g) In vitro treatment of HER2 + primary tumors cells treated with T-DM1 (5 nM) and assessed for sphere forming efficiency (black arrowhead). Below is shown immunoblotting of ROR1 from each group of patients' protein samples (results are mean +/− SE, n = 3 run in triplicate; Scale bar: 100 μM).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Expressing, Cell Culture, Staining, Immunofluorescence, Comparison, In Vitro, Western Blot

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: T-DM1 treatment alters ROR1 expression without significant changes in HER2 expression: (a) Immunostaining of HER2 expression from treatment naïve ( n = 5), sensitive (n = 5) and resistant (n = 5) HER2 + BC patient's tumor samples (Scale bar: 50 μM). (b) HCC1954 and MCF-7-HER2+ cells were cultured on a glass cover slip in 24-well plates and treated either with vehicle (DMSO) or 5 nM of T-DM1 for 5 days. Cells were fixed and immunofluorescence stained images captured for HER2 (red) and nucleus DAPI (blue) (Scale bar: 50 μM). (c) HCC1954 and MCF-7-HER2+ cells were cultured, treated and fixed as (b), and immunofluorescently stained for ROR1 (red) and nuclear DAPI (blue) expression (Scale bar: 50 μM). (d) Treatment naïve BC patient tumor cells, (e) HCC1954(HER2+), and (f) MCF-7-HER2+ cells were treated with T-DM1 (5 nM) for 5 days, trypsinized and cultured in supplemented CSC medium in sphere culture for 10 days (10 days is denoted as first generation). After 10 days, spheres were dissociated and re-cultured in CSC medium for an additional 10 days (20 days denoted as second generation) followed by dissociation of spheres and then re-cultured again in CSC medium (30 days denoted as third generation). In every 10-day culture cycle, sphere forming efficiency was examined in ROR1 + and ROR1 − populations by counting the number and size of spheres. (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = ns [not significant]; p = .05; p = .01, p = .001; one-way ANOVA). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Expressing, Immunostaining, Cell Culture, Immunofluorescence, Staining

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: T-DM1 treatment-induced ROR1 + cells enriched with CSC features show higher self-renewal efficiency and increased resistance with enriched CSC features. (a) Treatment naïve HER2 + BC patients tumor cells were enzymatically dissociated and cultured in supplemented CSC medium, treated with either vehicle (DMSO) and 5 nM T-DM1 and incubated for 10 days (follow the schematic diagram). Spheres were dissociated for single cells and subjected to FACS sorting for ROR1. Extreme limiting dilution assay (ELDA) was performed by plating sorted cells with increasing cell numbers and analyzed for stemness frequencies by ‘R' statistical package “statmod” ( p < .0427; one-way ANOVA). (right) showed the confidence interval for 1/stemness frequency. (b) Bulk cells were isolated and treated as described in (a) and analyzed for sphere forming efficiency (Bar graph represents the mean +/− SE, n = 3, run in triplicate). (c) HER2 + patient tumor cells treated with either vehicle or T-DM1 for 5 days, harvested and FACS sorted for ROR1 + and ROR1 − populations, and then sorted cells were cultured in supplemented CSC medium [follow the schematic diagram] for 10 days and analyzed for the sphere forming efficiency from each cell population (black arrowhead; p = .001 for T-DM1 treatment; p = .05 for vehicle, unpaired two-sided t -test, Scale bar: 100 μM). (d) HCC1954 cells were treated and analyzed as described in (c) [black arrowhead; p = .01 for T-DM1 treatment; p = ns (not significant) for vehicle, unpaired two-sided t -test; Scale bar: 100 μM]. (e) HCC1954 cells were either treated with vehicle or T-DM1 and sorted as described in (c) and quantitatively analyzed for Bmi1, Nanog, Oct3/4. Sox2 mRNA in ROR1 + and ROR1 − populations. (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05; p = .01; p = .001; one-way ANOVA). (f) ROR1 + cells were more significantly resistant than ROR1 − cells at individual T-DM1 treatment and showed higher EC50 in response to T-DM1 as compared to ROR1 − cells. (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05, p = .01).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Cell Culture, Incubation, Limiting Dilution Assay, Isolation

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: T-DM1 treatment induced ROR1 + cells are enriched for CD44, ALDH and YAP1 target genes: (a-b) Freshly dissociated treatment naïve and treatment resistant HER2 + BC patients tumor cells were FACS sorted and analyzed for ROR1 subpopulation stained with ALDH and CD44 antibodies (Results are median +/− interquartile range (IQR) n = 5; unpaired two-sided t -test). (c-d. Patients tumor cells were either treated with vehicle (DMSO) and/or T-DM1 for 5 days, and sorted as (a) Analysis of ALDH and CD44 expression by FACS and immunofluorescence staining in ROR1 + and ROR1 − subpopulation (Scale bar: 50 μM). (e) HCC1954 cells treated, maintained, sorted and analyzed as (d). (g) Cells were treated and sorted as (d), extracted total RNA followed by qRT-PCR for YAP1, TEAD1, CTGF and CCND1 in ROR1 + and ROR1 − populations (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .001, 0.01, 0.05, 0.001 and 0.001). (h) Detection of CTGF from treatment naïve and treatment exposed patients' blood serum by ELISA assay (Bar graph represents the mean +/− SE, n = 3; run in triplicate; ** p < .01; Students t -test).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Staining, Expressing, Immunofluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: Silencing ROR1 sensitizes T-DM1 treatment-induced ROR1 + spheroids forming efficiency and tumor initiation: (a) Patients tumor cells, (b) HCC1954, and (c) MCF-7 HER2+ cells. T-DM1 and ROR1-shRNA co-treatment resulted in eradication of sphere forming efficiency. (d-f) Loss of ROR1 expression in the spheres was analyzed by immunofluorescence staining (Scale bar: 50 μM) (red-ROR1, blue-DAPI) in HER2 + BC patients tumor cells, HCC1954 and MCF-7-HER2+ cells (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05; p = .01; one-way ANOVA). (g) Average number of spheres assessed for HCC1954 and MCF-7-HER2+ cells after transfecting with control shRNA and ROR1-shRNA. (h) Representation of tumors grown in mice after transfecting HCC1954 cells with either control-shRNA or ROR-shRNA. Table below indicates the number of tumors initiated in each group with percentage. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: shRNA, Expressing, Immunofluorescence, Staining, Control

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: T-DM1 treatment- induced ROR1 + cells have high tumor forming capacity in vivo : (a) Schematic representation of cell sorting and tumor initiation assay. (b) Fresh HER2 + breast tumor cells (n = 5 patient) were dissociated and FACS sorted ROR1 + and ROR1 − cells as described previously. Equal number of sorted and unsorted bulk cells from each patient tumor were injected subcutaneously in Nu/J mice (n = 5) and tumor growth and incidence were monitored for 8 weeks. ROR1 + cells were capable of generating larger tumors (5 out of 5 tumors), ROR1 − cells (2 out of 5 tumors) and unsorted bulk cells (3 out of 5 tumors) generated tumors but smaller in size (Scale bar 5 mm). (c) The weight distributions of tumors generated from ROR + , ROR1 − and unsorted bulk tumor cells are shown in all patient's tumors. Results are median +/− IQR (Inter quartile range), p = .01 and 0.05; repeated measure ANOVA. (d) HCC1954 cells were either treated with vehicle (DMSO) and/or T-DM1 for 5 days and sorted based on T-DM1 induced cell surface ROR1 overexpression and injected to mice. Tumor growth and incidence were monitored for 8 weeks. On the basis of tumor incidence in the indicated time, a Kaplan-Meier survival curve was generated using Log-rank test (Log-rank P = .03). “R” statistical software “survival” and “survminer” packages were used to analyze the tumor-free survival curve. (e) In vivo limiting dilution assay was performed using HCC1954 cells and treatment and sorting performed as described in (a). Sorted 100, 1000, 10,000 and 100,000 cells were mixed with Matrigel and injected subcutaneously in Nu/J mice (n = 5, Scale bar 5 mm). (f) Tumor initiating capacity was monitored over 8 weeks and CSC frequency was calculated using “R” statistical software “statmod” package. The frequency of tumor-initiating cells for 3 mice is graphed. Results are median +/− IQR (repeated measure ANOVA).

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: In Vivo, FACS, Injection, Generated, Over Expression, Software, Limiting Dilution Assay

Journal: EBioMedicine

Article Title: Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1

doi: 10.1016/j.ebiom.2019.04.061

Figure Lengend Snippet: YAP1 regulates T-DM1 treatment-induced ROR1 overexpression and CSC enrichment. (a) HCC1954 cells were transduced with full length YAP1 cDNA (PIN20YYAP1). Doxycycline (1 μg/ml) was added in the cell culture medium to induce the YAP1 expression. Western blot was performed using antibodies to YAP1, ROR1 and HER2. (b) Immunofluorescence staining for YAP1 (green; white arrow head) and ROR1 (red; white arrowhead) and DAPI (blue) confirming the transduction of YAP1 in the presence and absence of doxycycline (Scale bar: 50 μM). (c) YAP1 was knockdown by two independent YAP1 siRNA and Western Blot was performed to confirm the expression of YAP1 and ROR1 (red label box siRNA was used in our experiments). (d) HCC1954 cells were transfected with non-targeting and YAP1 specific siRNA for 48 h. Cells were then treated with T-DM1 for 48 h, followed by treatment with ROR1-shRNA for 48 h. Treated cells were trypsinized, cultured in the supplemented CSC medium, and assessed for sphere forming efficiency (black arrowhead) Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05; p = .01; one-way ANOVA. [Scale bar: 100 μM]. (e) Immunoblots showing the expression of YAP1, ROR1 and HER2 after treatment of HCC1954 cells with 2 μg/mL verteporfin. (f) Quantification of sphere forming efficiency (black arrowhead) after verteporfin treatment at 2 μg/ml and vehicle (DMSO) in HCC1954 cells. Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05; p = .01; Student's t -test; Scale bar: 100 μM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human ROR1 (AF2000, R&D System, USA), HER2 (MAB1129, R & D System, USA) and YAP1(MAB8094, R & D System, USA) antibodies were purchased from R & D System, USA.

Techniques: Over Expression, Transduction, Cell Culture, Expressing, Western Blot, Immunofluorescence, Staining, Knockdown, Transfection, shRNA

Journal: Life

Article Title: A Histological Analysis and Detection of Complement Regulatory Protein CD55 in SARS-CoV-2 Infected Lungs

doi: 10.3390/life14091058

Figure Lengend Snippet: Representative overlay immunofluorescence images of lung tissues derived from COVID–/ARDS–, COVID+/ARDS+, and COVID–/ARDS+ patients in 200× (upper row: ( A , C , E , G ). Scalebar: 100 µm) and 630× (lower row: ( B , D , F ). Scalebar: 20 µm) magnification. Red (Cy3) = CD55, blue (DAPI) = cell nuclei. The primary antibody against CD55 was omitted in the negative control. ( A ) The blood vessel inside the lung tissues has been indicated by the oval lining. The yellow arrow indicates a staining artifact. The white arrows in image ( B ) depict alveolar macrophages with positive CD55 staining. ( H ) Graphic representation of relative CD55 protein fluorescence intensity measured in [COVID–/ARDS–, n = 4], [COVID+/ARDS+, n = 5] and [COVID–/ARDS+, n = 3], lung tissues. Mean with standard deviation (SD). The COVID–/ARDS– group has been normalized to 100. Repeated Measures one-way ANOVA using Dunnett’s multiple comparisons (*). * = p ≤ 0.05.

Article Snippet: For antigen retrieval, the slides were treated in a pressure cooker (Rommelbacher ElektroHausgeräte GmbH, Dinkelsbühl, Germany) at 97 °C, pH 6, and steam pressure of 1 bar for 10 min. After the antigen retrieval procedure and cooling of the slides upto RT, the slides were first rinsed in PBS for 5 min before the primary antibody, CD55 (Antigen Affinity-purified Polyclonal Goat IgG, Catalog Number: AF2009, R&D systems, Minneapolis, MN, USA) [ ] diluted in blocking solution was pipetted onto the slides and incubated at 4 °C overnight.

Techniques: Immunofluorescence, Derivative Assay, Negative Control, Staining, Fluorescence, Standard Deviation

Journal: Life

Article Title: A Histological Analysis and Detection of Complement Regulatory Protein CD55 in SARS-CoV-2 Infected Lungs

doi: 10.3390/life14091058

Figure Lengend Snippet: ( A ) A representative overlay immunofluorescence image of COVID–/ARDS– lung tissue containing a vessel with circa 200 µm thick muscle layer (double-headed red arrow). Red (Cy3) = CD55, blue (DAPI) = cell nuclei. ( B ) Image A in the processing sequence of the LASX software analysis tool (Version 3.5.7.23225) containing blue as well as red channels, ( C ) single red channel extraction from image B, ( D ) a binary image derived from image C. Scalebar 100 µm.

Article Snippet: For antigen retrieval, the slides were treated in a pressure cooker (Rommelbacher ElektroHausgeräte GmbH, Dinkelsbühl, Germany) at 97 °C, pH 6, and steam pressure of 1 bar for 10 min. After the antigen retrieval procedure and cooling of the slides upto RT, the slides were first rinsed in PBS for 5 min before the primary antibody, CD55 (Antigen Affinity-purified Polyclonal Goat IgG, Catalog Number: AF2009, R&D systems, Minneapolis, MN, USA) [ ] diluted in blocking solution was pipetted onto the slides and incubated at 4 °C overnight.

Techniques: Immunofluorescence, Sequencing, Software, Extraction, Derivative Assay