Journal: Oncotarget

Article Title: Immunotherapy of ovarian cancer with a monoclonal antibody specific for the extracellular domain of anti-Müllerian hormone receptor II

doi: 10.18632/oncotarget.27585

Figure Lengend Snippet: A panel of approximately 300 hybridoma supernatants were generated and screened for specificity against rhAMHR2-ED and the 4D12 parental hybridoma was selected for subcloning by limiting dilution. ( A ) Subcloning produced three sub-clones, 4D12C6, 4D12C7, and 4D12G1 each of which expressed the IgG 1 /κ-chain isotype and showed antigen specificity ( B ) by competitive ELISA and ( C ) by flow cytometry binding to OVCAR8 cells. For flow cytometry, positive control staining of OVCAR8 cells was performed using a commercially available anti-AMHR2-ED mAb (Abcam), whereas IgG1 isotype antibodies with irrelevant specificities were used as negative controls. In all cases, error bars indicate ± SD and the results shown are representative of three experiments yielding similar results.

Article Snippet: The AMHR2-OVCAR8 cell line was generated by cloning transcript variant 1 of human AMHR2 (Accession #NM_020547; Origene, Rockville, MD) into pEZ-M68 vector (GeneCopoeia, Rockville, MD) and using this expression vector to transfect OVCAR8 cells using Lipofectamine™ 3000 (Thermo Fisher).

Techniques: Generated, Subcloning, Produced, Clone Assay, Competitive ELISA, Flow Cytometry, Binding Assay, Positive Control, Staining

Journal: Oncotarget

Article Title: Immunotherapy of ovarian cancer with a monoclonal antibody specific for the extracellular domain of anti-Müllerian hormone receptor II

doi: 10.18632/oncotarget.27585

Figure Lengend Snippet: ( A ) Flow cytometry analysis showing that the 4D12G1 mAb binds to the majority of cells generated from two primary HGSOC tissues examined. Error bars indicate ± SD. ( B ) The 4D12G1 mAb was used in Western blots of seven different HGSOC tissue lysates (25 μg protein/lane) with a positive control lysate generated from a young C57BL/6 ovary and a negative control lysate generated from C4-2 human prostate cancer cells. Immunostaining with a β-actin antibody was used to confirm normalized lysate loading. The Western blots shown are representative of three experiments that provided similar results. ( C ) The 4D12G1 mAb was used in immunohistochemical staining (20 ×) of tissue sections from four HGSOC patients (left column) and their normal adjacent fallopian tube tissues (right column). Arrows indicate staining of the tumor parenchyma. The stromal areas of the EOC tumors were not immunostained nor were all areas of the normal adjacent fallopian tube tissues. All experiments were performed three times yielding similar results. ( D ) Western blot analysis of lysates from OVCAR8 cells and AMHR2-OVCAR8 cells with lysates from C4-2 prostate cancer cells used as controls and immunostaining with a β-actin antibody was used to confirm normalized lysate loading. Flow cytometry analysis showed that: ( E ) the 4D12G1 mAb binds to 91% of AMHR2-OVCAR8 cells; ( F ) the AMH cognate ligand for AMHR2-ED effectively competes in a dose-dependent manner with the 4D12G1 mAb for binding to AMHR2-OVCAR8 cells; and ( G ) recombinant ovalbumin failed to compete with the 4D12G1 mAb for binding to AMHR2-OVCAR8 cells. Data are representative of three independent experiments yielding similar results.

Article Snippet: The AMHR2-OVCAR8 cell line was generated by cloning transcript variant 1 of human AMHR2 (Accession #NM_020547; Origene, Rockville, MD) into pEZ-M68 vector (GeneCopoeia, Rockville, MD) and using this expression vector to transfect OVCAR8 cells using Lipofectamine™ 3000 (Thermo Fisher).

Techniques: Flow Cytometry, Generated, Western Blot, Positive Control, Negative Control, Immunostaining, Immunohistochemical staining, Staining, Binding Assay, Recombinant

Journal: Oncotarget

Article Title: Immunotherapy of ovarian cancer with a monoclonal antibody specific for the extracellular domain of anti-Müllerian hormone receptor II

doi: 10.18632/oncotarget.27585

Figure Lengend Snippet: Details of EOC patients and their examined tumors

Article Snippet: The AMHR2-OVCAR8 cell line was generated by cloning transcript variant 1 of human AMHR2 (Accession #NM_020547; Origene, Rockville, MD) into pEZ-M68 vector (GeneCopoeia, Rockville, MD) and using this expression vector to transfect OVCAR8 cells using Lipofectamine™ 3000 (Thermo Fisher).

Techniques: Immunohistochemical staining, Expressing

Journal: Oncotarget

Article Title: Immunotherapy of ovarian cancer with a monoclonal antibody specific for the extracellular domain of anti-Müllerian hormone receptor II

doi: 10.18632/oncotarget.27585

Figure Lengend Snippet: ( A ) The entire 132 amino acid sequence of human AMHR2-ED. ( B ) An overlapping series of 16-mer peptides spanning the entire sequence of human AMHR2-ED with one amino acid shifts were plated for direct ELISA testing using the 4D12G1 mAb as the primary antibody. The 4D12G1 mAb recognized residues AMHR2-ED 11–32. ( C ) Overlapping peptides spanning AMHR2-ED 13–30 were synthesized with alanine substitutions at each N-terminal residue or with glycine substitutions for any native N-terminal alanine residues. Competitive ELISA results showed that alanine substitutions at residues spanning AMHR2-ED 20-26 ( 20 KTLGELL 26 ) decreased binding of the 4D12G1 mAb to AMHR2-ED. ( D ) SPOT peptide arrays using 4-16-mer peptides spanning AMHR2-ED 9-40 were immobilized on cellulose membranes, treated with the 4D12G1 mAb, and the bound antibody was detected by chemiluminescence. The results showed that the AMHR2-ED 22–26 5-mer sequence ( 22 LGELL 26 ) represents the minimal sequence for binding of the 4D12G1 mAb. ( E ) SPOT peptide arrays were made using membrane bound 17-mer peptides spanning the AMHR2-ED 17–33 domain and containing alanine substitutions at each sequential amino acid. Alanine replacement of Leu 22 , Gly 23 , and Leu 26 completely abolished binding by the 4D12G1 mAb. All error bars indicate ±SD, and all experiments are representative of three experiments yielding similar data.

Article Snippet: The AMHR2-OVCAR8 cell line was generated by cloning transcript variant 1 of human AMHR2 (Accession #NM_020547; Origene, Rockville, MD) into pEZ-M68 vector (GeneCopoeia, Rockville, MD) and using this expression vector to transfect OVCAR8 cells using Lipofectamine™ 3000 (Thermo Fisher).

Techniques: Sequencing, Direct ELISA, Synthesized, Competitive ELISA, Binding Assay

Journal: Oncotarget

Article Title: Immunotherapy of ovarian cancer with a monoclonal antibody specific for the extracellular domain of anti-Müllerian hormone receptor II

doi: 10.18632/oncotarget.27585

Figure Lengend Snippet: ( A ) AMHR2-OVCAR8 cells were treated with a green fluorescent dye and the 4D12G1 mAb or an isotype control mAb. Apoptosis was assessed by live imaging using the IncuCyte S3 analyzer. 4D12G1 mAb induced substantial apoptosis at 16 hours (right panel) compared to isotype control mAb (left panel). ( B ) AMHR2-OVCAR8 cells were treated with different concentrations of the 4D12G1 mAb for 24 hours and Western blots of the cell lysates showed detection of the intact 116 kDa PARP-1 and its 89 kDa cleaved variant, consistent with apoptosis. Immunostaining with a β-actin antibody was used to confirm normalized lysate loading. ( C ) AMHR2-OVCAR8 cells were incubated with the 4D12G1 mAb for different time periods at either 37° C (left column) or 4° C (right column). Clustered patterns of cytoplasmic antibody-receptor complexes became increasingly more prominent at 2 and 3 hours after treatment at 37° C, but not at 4° C, and no staining occurred in cells treated with secondary antibody alone (right column, bottom panel). ( D ) AMHR2-OVCAR8 cells were incubated in either 10% normal human serum or 10% heat-inactivated human serum and treated for 4 hours with varying doses of either 4D12G1 mAb or isotype control mAb. Cell lysis mediated by CDC was measured by release of LDH activity and occurred only in cells treated with the 4D12G1 mAb. ( E ) AMHR2-OVCAR8 target cells were labeled with a green fluorescent dye and incubated with two different concentrations of 4D12G1 mAb or isotype control mAb. The cells were mixed with effector macrophages from C57BL/6 mouse bone marrow at an effector to target cell ratio of 10:1. Live target cells were analyzed by flow cytometry 3 days later for demonstrating ADCP. All error bars indicate ±SD. All experiments are representative of three experiments yielding similar data.

Article Snippet: The AMHR2-OVCAR8 cell line was generated by cloning transcript variant 1 of human AMHR2 (Accession #NM_020547; Origene, Rockville, MD) into pEZ-M68 vector (GeneCopoeia, Rockville, MD) and using this expression vector to transfect OVCAR8 cells using Lipofectamine™ 3000 (Thermo Fisher).

Techniques: Imaging, Western Blot, Variant Assay, Immunostaining, Incubation, Staining, Lysis, Activity Assay, Labeling, Flow Cytometry

Journal: Oncotarget

Article Title: Immunotherapy of ovarian cancer with a monoclonal antibody specific for the extracellular domain of anti-Müllerian hormone receptor II

doi: 10.18632/oncotarget.27585

Figure Lengend Snippet: Human EOC tumors were injected s. c. into immunodeficient mice. When tumors became palpable, mice were injected i. p. with 200 μg of either the 4D12G1 mAb or an isotype control mAb weekly for 5 continuous weeks. Treatment with the 4D12G1 mAb significantly inhibited the growth of OVCAR8 tumors in ( A ) severely immunodeficient NSG mice ( P < 0.001) and in ( B ) T cell-deficient athymic nude mice ( P < 0.0001). More importantly, treatment with the 4D12G1 mAb significantly inhibited the growth of three primary HGSOC tumors ( P < 0.0001 in all cases) generated from recently diagnosed patients and xenografted into immunodeficient NSG mice including ( C ) PDX-4, ( D ) PDX-6, and ( E ) PDX-9. ( F ) Detection of caspase-3 positive cells in the OVCAR8 (upper row) and PDX-4 tumors (lower row) from NSG mice at 20× is shown by arrows in mice treated with the 4D12G1 mAb (right column) compared to mice treated with isotype control mAb (left column). Caspase-3 data shown are representative of three experiments yielding similar results. All error bars indicate ± SD.

Article Snippet: The AMHR2-OVCAR8 cell line was generated by cloning transcript variant 1 of human AMHR2 (Accession #NM_020547; Origene, Rockville, MD) into pEZ-M68 vector (GeneCopoeia, Rockville, MD) and using this expression vector to transfect OVCAR8 cells using Lipofectamine™ 3000 (Thermo Fisher).

Techniques: Injection, Generated

Journal: Scientific Reports

Article Title: Targeted therapy of pyrrolo[2,3- d ]pyrimidine antifolates in a syngeneic mouse model of high grade serous ovarian cancer and the impact on the tumor microenvironment

doi: 10.1038/s41598-022-14788-5

Figure Lengend Snippet: Expression of GARFTase and AICARFTase transcripts in primary EOC patient samples and BR-5 and BR-Luc cells. Transcript levels for GARFTase ( A ) and AICARFTase ( B ) were measured using cDNAs from primary specimens including normal ovary (n = 8) and HGSOC (n = 39) (OriGene) and results were compared to those for EOC cell lines including IGROV1, SKOV3, A2780 and A2780 E-80. Transcript levels were normalized to β-actin transcripts. Statistical analyses were performed between normal samples/tissues and tumor samples/tissues using the Wilcoxon rank-sum test. ( C , D ) Transcript levels of cytosolic C1 metabolic targets (GARFTase and AICARFTase) were determined in the BR-5 and BR-Luc syngeneic mouse models of HGSOC by real-time RT-PCR. Transcripts were normalized to β-actin transcripts and results are shown relative to levels in mouse liver (assigned a value of 1). Results are presented as mean values ± standard errors from at least three experiments. The p values are as follows: **** p < 0.0001. See Supplementary Table for patient characteristics and pathology.

Article Snippet: Transcript levels for GARFTase ( A ) and AICARFTase ( B ) were measured using cDNAs from primary specimens including normal ovary (n = 8) and HGSOC (n = 39) (OriGene) and results were compared to those for EOC cell lines including IGROV1, SKOV3, A2780 and A2780 E-80.

Techniques: Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Targeted therapy of pyrrolo[2,3- d ]pyrimidine antifolates in a syngeneic mouse model of high grade serous ovarian cancer and the impact on the tumor microenvironment

doi: 10.1038/s41598-022-14788-5

Figure Lengend Snippet: IC 50 values (in nM) for anti-proliferative activities of novel compounds on HGSOC mouse models BR-Luc and BR-5 compared to cisplatin.

Article Snippet: Transcript levels for GARFTase ( A ) and AICARFTase ( B ) were measured using cDNAs from primary specimens including normal ovary (n = 8) and HGSOC (n = 39) (OriGene) and results were compared to those for EOC cell lines including IGROV1, SKOV3, A2780 and A2780 E-80.

Techniques:

Journal: Cancer Cell International

Article Title: Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer

doi: 10.1186/s12935-022-02838-x

Figure Lengend Snippet: siRNA suppression of TIMP-2 in the OVCAR5 cell line. Suppression of TIMP-2 expression by siRNA transfection in the OVCAR5 cell line is described in “ ”. TIMP-2 expression was evaluated by immunofluorescence at the protein level and at mRNA level by qRT-PCR as described in “ ”. A, B and C are single siRNA duplexes and A + B + C is representative of a pool of all three TIMP-2 siRNAs at a 3 nM final concentration. Immunofluorescence images are representation of merged DAPI (blue) and TIMP-2 (red) staining on individual cell lines done in three passages in triplicate. The intensity of fluorescence was obtained using FIJI software. ×20 magnification; scale bar (in yellow) 20 μM; P indicates the parental cell line treated with transfection reagent only, Cont are cells transfected with scrambled siRNA. For mRNA expression, graphs represent amount of mRNA relative to 18S + SEM derived from three experiments done in triplicate. Significance was determined by one-way ANOVA and indicated by ***p < 0.001, ****p < 0.0001

Article Snippet: The OVCAR5 cell line was co-transfected with a gRNA plasmid and donor fragment [TIMP-2 Human Gene Knockout Kit (CRISPR), KN209796, Origene Rockville, Maryland, US].

Techniques: Expressing, Transfection, Immunofluorescence, Quantitative RT-PCR, Concentration Assay, Staining, Fluorescence, Software, Derivative Assay

Journal: Cancer Cell International

Article Title: Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer

doi: 10.1186/s12935-022-02838-x

Figure Lengend Snippet: Expression of TIMP-2 and other TIMPs in response to TIMP-2 suppression by CRISPR/Cas9 and siRNA in OVCAR5 cell line. Cell lines studied are P—Parental; C—CRISPR control; 2—gRNA2, 1—gRNA1 cell lines; Cont-siRNA control, T2-KD-TIMP-2 siRNA knocked down and P, parental lipofectamine only treated control cells. A Expression of cellular TIMP-2 by Western blot in parental and CRISPR/Cas9 treated cell lines. Representative image of a Western blot of TIMP-2 and GAPDH proteins on the cell lysates of the respective cell lines. The additional cross-reactive bands observed are indicated in manufacture’s information. Graphs indicates densitometry intensity units of TIMP-2 protein bands normalized to GAPDH protein. The data presented is a mean of three independent experiments. Values are mean ± SEM. Significance was obtained using One-way ANOVA. **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared to the control and parental cell lines. B Expression of cellular TIMP-2 by immunofluorescence in CRISPR/Cas9 treated, control and parental cell lines. Images are representations of merged DAPI (blue) and TIMP-2 (red) staining of individual cell lines performed in three independent experiments, in triplicate. ×40 magnification; scale bar (in yellow) 40 µM. Graphs indicates the intensity of fluorescence obtained using FIJI software as described in “ ”. Significance was obtained using One-way ANOVA. C Secreted TIMP-2 protein in CRISPR/Cas9 treated, control and parental cell lines. The expression of secreted TIMP-2 in cell conditioned medium was deduced by ELISA as described in “ ”. Results are expressed as total TIMP-2 secreted protein (pg/µg of protein in cell medium). Values are mean ± SEM and each bar graph represents n = 3 for each cell line collected in three independent experiments. Significance was obtained using One-way ANOVA *p < 0.05, **p < 0.01, and ****p < 0.0001 when compared to the control cell line and parental cell line. D , E mRNA expression of TIMP-1 and TIMP-3 in siRNA-mediated TIMP-2 suppressed cells. mRNA expression of TIMP-1 and TIMP-3 was deduced by qRT-PCR as described in “ ”. Values are mean ± SEM and each bar graph represents n = 3 for each cell line collected in three independent experiments

Article Snippet: The OVCAR5 cell line was co-transfected with a gRNA plasmid and donor fragment [TIMP-2 Human Gene Knockout Kit (CRISPR), KN209796, Origene Rockville, Maryland, US].

Techniques: Expressing, CRISPR, Western Blot, Immunofluorescence, Staining, Fluorescence, Software, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Cancer Cell International

Article Title: Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer

doi: 10.1186/s12935-022-02838-x

Figure Lengend Snippet: Effect of TIMP-2 suppression by A CRISPR/Cas9 and B siRNA methods on the mRNA expression of EMT-associated genes in OVCAR5 cells. The cell lines/cells are described in Fig. . A , B The mRNA expression of E-Cad, N-Cad, VIM, SLUG, TWIST1, SNAIL and TGFβ1 deduced by qRT-PCR. Values are mean ± SEM and each bar graph represents n = 3 for each cell line collected in independent experiments. Significance was obtained using One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 when compared to the control and parental cell lines

Article Snippet: The OVCAR5 cell line was co-transfected with a gRNA plasmid and donor fragment [TIMP-2 Human Gene Knockout Kit (CRISPR), KN209796, Origene Rockville, Maryland, US].

Techniques: CRISPR, Expressing, Quantitative RT-PCR

Journal: Cancer Cell International

Article Title: Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer

doi: 10.1186/s12935-022-02838-x

Figure Lengend Snippet: An in vitro and in vivo summary of the effects of disruption of TIMP-2 expression by either siRNA or CRISPR/Cas9 in OVCAR5 ovarian cancer cell line. Suppression of TIMP-2 expression by 60–76% in T2-KD cells and gRNA2 cell line resulted in EMT-associated changes (decreased expression of E-cad and MMP-2 with corresponding increased expression of MMP-14, SLUG, SNAIL, VIM, N-cad and TGFβ1). There was also increased proliferation, invasion and chemosensitivity to PTX. In 3D spheroid cultures, gRNA2 cells formed tight, round, and compact cell aggregates, that overexpressed MMP-2, MMP-14, E-Cad, N-Cad and TGFβ1. However, when injected into mice they produced a small tumour burden with no tumours infiltrating peritoneal organs and the mice had higher survival rates compared to control and parental cell lines. On the other hand, in gRNA1, edited by CRISPR/Cas9 for TIMP-2 gene, resulted in the inhibition of TIMP-2 expression by 81–87%. These cells exhibited low MMP-2 expression, and high MMP-14, TWIST1 and SNAIL expression, enhanced proliferation, migration, and invasion compared to control cell lines. However, this cell line was resistant to PTX and in 3D spheroid cultures formed long sheath-like cell aggregates, that had enhanced proliferation and expression of invasion marker, KRT14. Furthermore, when injected into mice gRNA1 cells produced a high tumour burden, which infiltrated peritoneal organs such as liver and pancreas and had similar survival rates when compared to controls. These data suggests that the differences in the ratios of TIMP-2 and MMPs may be critical in controlling the tumorigenic functions of ovarian cancer cells

Article Snippet: The OVCAR5 cell line was co-transfected with a gRNA plasmid and donor fragment [TIMP-2 Human Gene Knockout Kit (CRISPR), KN209796, Origene Rockville, Maryland, US].

Techniques: In Vitro, In Vivo, Expressing, CRISPR, Injection, Produced, Inhibition, Migration, Marker

Journal: Scientific Reports

Article Title: Targeted therapy of pyrrolo[2,3- d ]pyrimidine antifolates in a syngeneic mouse model of high grade serous ovarian cancer and the impact on the tumor microenvironment

doi: 10.1038/s41598-022-14788-5

Figure Lengend Snippet: Expression of GARFTase and AICARFTase transcripts in primary EOC patient samples and BR-5 and BR-Luc cells. Transcript levels for GARFTase ( A ) and AICARFTase ( B ) were measured using cDNAs from primary specimens including normal ovary (n = 8) and HGSOC (n = 39) (OriGene) and results were compared to those for EOC cell lines including IGROV1, SKOV3, A2780 and A2780 E-80. Transcript levels were normalized to β-actin transcripts. Statistical analyses were performed between normal samples/tissues and tumor samples/tissues using the Wilcoxon rank-sum test. ( C , D ) Transcript levels of cytosolic C1 metabolic targets (GARFTase and AICARFTase) were determined in the BR-5 and BR-Luc syngeneic mouse models of HGSOC by real-time RT-PCR. Transcripts were normalized to β-actin transcripts and results are shown relative to levels in mouse liver (assigned a value of 1). Results are presented as mean values ± standard errors from at least three experiments. The p values are as follows: **** p < 0.0001. See Supplementary Table for patient characteristics and pathology.

Article Snippet: Transcript levels for GARFTase ( A ) and AICARFTase ( B ) were measured using cDNAs from primary specimens including normal ovary (n = 8) and HGSOC (n = 39) (OriGene) and results were compared to those for EOC cell lines including IGROV1, SKOV3, A2780 and A2780 E-80.

Techniques: Expressing, Quantitative RT-PCR