Journal: Molecular Therapy

Article Title: VEGF Blockade Enables Oncolytic Cancer Virotherapy in Part by Modulating Intratumoral Myeloid Cells

doi: 10.1038/mt.2013.39

Figure Lengend Snippet: Analysis of rRp450 efficacy in sarcoma models. (a) Tumor cells were infected with rRp450 at the indicated MOI and harvested for HSV titer as determined by standard plaque assay at 1, 24, 48, and 72 hours post-infection (n = 4). Error bars represent SEM. (b) A673 cells were infected at the indicated MOI and cell viability was measured on days 2, 4, and 6 by MTT assay (n = 4). Error bars represent SD. (c) Mice bearing A673 tumors received two intratumoral injections of rRp450 at 1 × 107 pfu or PBS control, on days 0 and 2, and then followed for tumor growth (n = 7). (d) Mice bearing 143.98.2 tumors received two intratumoral injections of rRp450 at 1 × 107 pfu or PBS as a control on days 0 and 2, and were followed for tumor growth (n = 5–10). CR, complete response; ITu, intratumoral; MOI, multiplicity of infection; oHSV, oncolytic herpes simplex virus; PBS, phosphate-buffered saline; pfu, plaque-forming unit; PD, progressive disease; PR, partial response; SD, stable disease.

Article Snippet: Six to eight weeks old female athymic nude (nu/nu) mice (Harlan Sprague Dawley, Indianapolis, IN) were injected subcutaneously with A673 Ewing sarcoma cells (5 × 10 6 ) in 30% matrigel (BD Biosciences, San Diego, CA).

Techniques: Infection, Plaque Assay, MTT Assay

Journal: Molecular Therapy

Article Title: VEGF Blockade Enables Oncolytic Cancer Virotherapy in Part by Modulating Intratumoral Myeloid Cells

doi: 10.1038/mt.2013.39

Figure Lengend Snippet: Recruitment of myeloid cells in oHSV-injected tumors. A673 xenograft tumors were injected with rRp450 or PBS control. (a) The relative numbers of CD11b+ myeloid cells in the spleen (n = 4–5, t-test) and infected flank tumors (n = 6, t-test) were determined 3 days after virus injection by flow cytometry. In a separate experiment, (b,c) spleens and (d,e) tumors were collected 24 hours after virus injection and analyzed by hematoxylin and eosin (H&E) and by Ly6G immunohistochemistry staining (4X objective). The spleen served as a control, and brown staining is restricted to the red pulp and absent from the white pulp. oHSV, oncolytic herpes simplex virus; PBS, phosphate-­buffered saline.

Article Snippet: Six to eight weeks old female athymic nude (nu/nu) mice (Harlan Sprague Dawley, Indianapolis, IN) were injected subcutaneously with A673 Ewing sarcoma cells (5 × 10 6 ) in 30% matrigel (BD Biosciences, San Diego, CA).

Techniques: Injection, Infection, Flow Cytometry, Immunohistochemistry, Staining

Journal: Molecular Therapy

Article Title: VEGF Blockade Enables Oncolytic Cancer Virotherapy in Part by Modulating Intratumoral Myeloid Cells

doi: 10.1038/mt.2013.39

Figure Lengend Snippet: Induction of mVEGF in oHSV-injected tumors. A673 xenograft tumors were injected with rRp450 or PBS control and tumors were harvested at 3 days. The amount of (a) tumor-derived hVEGF (n = 7) and (b) stroma-derived mVEGF was determined by ELISA (n = 7). Error bars represent SEM. ELISA, enzyme-linked immunosorbent assay; hVEGF, human vascular endothelial growth factor; mVEGF, mouse vascular endothelial growth factor; oHSV, oncolytic herpes simplex virus; PBS, phosphate-buffered saline.

Article Snippet: Six to eight weeks old female athymic nude (nu/nu) mice (Harlan Sprague Dawley, Indianapolis, IN) were injected subcutaneously with A673 Ewing sarcoma cells (5 × 10 6 ) in 30% matrigel (BD Biosciences, San Diego, CA).

Techniques: Injection, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy

Article Title: VEGF Blockade Enables Oncolytic Cancer Virotherapy in Part by Modulating Intratumoral Myeloid Cells

doi: 10.1038/mt.2013.39

Figure Lengend Snippet: Depletion of CD11b+ cells in oHSV-injected tumors. (a) A673 tumor-bearing animals were administered intraperitoneally either αβ-gal antibody (control) or α-CD11b antibody on the indicated days and either intratumoral PBS (control) or rRp450 (oHSV) on day 0. (b) Tumors were collected at day +3 and analyzed by flow cytometry. Shown are representative scatter plots from an n = 3 experiment illustrating the baseline CD11b+ and GR1+ populations, their depletion by anti-CD11b antibody injection, and their increase with oHSV infection. Only one scatter plot is shown for CD11b depletions because all six were essentially identical, with absence of CD11b cells regardless of PBS or oHSV injection. (c) Tumors were analyzed by ELISA for murine VEGF production (n = 5–8). Error bars represent SEM. ELISA, enzyme-linked immunosorbent assay; IP, intraperitoneal; IT, intratumoral; oHSV, oncolytic herpes simplex virus; PBS, phosphate-buffered saline; VEGF, vascular endothelial growth factor.

Article Snippet: Six to eight weeks old female athymic nude (nu/nu) mice (Harlan Sprague Dawley, Indianapolis, IN) were injected subcutaneously with A673 Ewing sarcoma cells (5 × 10 6 ) in 30% matrigel (BD Biosciences, San Diego, CA).

Techniques: Injection, Flow Cytometry, Infection, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy

Article Title: VEGF Blockade Enables Oncolytic Cancer Virotherapy in Part by Modulating Intratumoral Myeloid Cells

doi: 10.1038/mt.2013.39

Figure Lengend Snippet: Treatment of A673 xenografts with combination bevacizumab and oHSV. Mice bearing A673 xenografts were treated with ITu oHSV, IP bevacizumab or a combination of both and followed for (a) tumor growth (n = 7–8) and (b) survival (n = 7–8). Error bars represent SEM. (c) Intratumoral virus production was measured in a separate cohort. Tumors were harvested at times shown and measured for infectious virus particles by plaque assay. Bevacizumab reduced virus production in the tumors, likely due to the antiangiogenic effects causing tumor cell death and limiting virus spread and production (n = 6). Error bars represent SEM. Bev, bevacizumab; IP, intraperitoneal; ITu, intratumoral; oHSV, oncolytic herpes simplex virus; pfu, plaque-forming unit.

Article Snippet: Six to eight weeks old female athymic nude (nu/nu) mice (Harlan Sprague Dawley, Indianapolis, IN) were injected subcutaneously with A673 Ewing sarcoma cells (5 × 10 6 ) in 30% matrigel (BD Biosciences, San Diego, CA).

Techniques: Plaque Assay

Journal: Molecular Therapy

Article Title: VEGF Blockade Enables Oncolytic Cancer Virotherapy in Part by Modulating Intratumoral Myeloid Cells

doi: 10.1038/mt.2013.39

Figure Lengend Snippet: Effects of the combination of intratumoral oHSV and intraperitoneal bevacizumab on the tumor vasculature. A673 tumor-bearing animals were administered either ITu PBS and IP rat IgG control antibody, ITu rRp450 and IP rat IgG control antibody, ITu PBS and IP bevacizumab or ITu rRp450 and IP bevacizumab. Tumors were harvested 3 days post-infection and either analyzed by ELISA for (a) hVEGF (n = 4–5) and (b) mVEGF (n = 4–6), evaluated by immunohistochemistry for apoptosis by TUNEL staining and for (c) pericytes by α-smooth muscle actin (α-SMA) staining or (d) endothelial cells by CD31 staining. Error bars represent SD (in a,b). Tumors were also quantified for (e) vessel numbers (n = 6, 10 high power fields per tumor) and (f) microvessel density (total vessel area per high power field; n = 6, 10 high power fields per tumor). Error bars represent SEM. Data were compared with PBS and only those indicated by an asterisk reached statistical significance. Bev, bevacizumab; ELISA, enzyme-linked immunosorbent assay; hpf, high power field; hVEGF, human vascular endothelial growth factor; mVEGF, mouse vascular endothelial growth factor; IP, intraperitoneal; ITu, intratumoral; oHSV, oncolytic herpes simplex virus; PBS, phosphate-buffered saline; TUNEL, terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling.

Article Snippet: Six to eight weeks old female athymic nude (nu/nu) mice (Harlan Sprague Dawley, Indianapolis, IN) were injected subcutaneously with A673 Ewing sarcoma cells (5 × 10 6 ) in 30% matrigel (BD Biosciences, San Diego, CA).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, TUNEL Assay, Staining, End Labeling

Journal: Molecular Therapy

Article Title: VEGF Blockade Enables Oncolytic Cancer Virotherapy in Part by Modulating Intratumoral Myeloid Cells

doi: 10.1038/mt.2013.39

Figure Lengend Snippet: Model of enhancement of oHSV efficacy by VEGF blockade. A model consistent with our data is that virus infection of tumor cells stimulates an innate neutrophilic infiltration and, through an unknown mechanism, depletes M1-type tumor-associated macrophages. Infection also induces production of host-derived mVEGF, either directly or indirectly via myeloid cells. The increase in stroma-derived mVEGF is an example of the virus effects on the local tumor microenvironment, but in the A673 model is overshadowed by tumor-derived hVEGF and likely plays only a minor role in angiogenesis. (We postulate that the effect of stroma-derived mVEGF may be more impactful in models with less tumor-derived hVEGF production). The virus-induced CD11b+ infiltrate results in production of protumor growth and proangiogenic factors from neutrophils and M2-type macrophages (+ factors represented by small arrows) that are no longer offset by M1-type macrophages. The combination of oHSV and anti-VEGF is more antiangiogenic, and the resulting tumor cell death is likely responsible for decreased intratumoral virus spread and production. Despite lower virus production, the combination results in improved antitumor effects due in part to modulation of the intratumoral myeloid cell composition, specifically by a mitigation of the decrease in tumoricidal M1-type macrophages. These effects are dependent on VEGFR2 signaling as they were seen with bevacizumab and r84. Modulation of myeloid cells is in part responsible for the combined effects of oHSV with VEGF blockade as it could be recapitulated by combining oHSV with depletion of CD11b cells, possibly by preventing a virus-induced predominance of M2-type compared with M1-type macrophages. The "?" denotes unknown mechanism. Bev, bevacizumab; hVEGF, human vascular endothelial growth factor; mVEGF, mouse vascular endothelial growth factor; oHSV, oncolytic herpes simplex virus; VEGFR, vascular endothelial growth ­factor receptor.

Article Snippet: Six to eight weeks old female athymic nude (nu/nu) mice (Harlan Sprague Dawley, Indianapolis, IN) were injected subcutaneously with A673 Ewing sarcoma cells (5 × 10 6 ) in 30% matrigel (BD Biosciences, San Diego, CA).

Techniques: Infection, Derivative Assay

Journal: Cell Death & Disease

Article Title: SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

doi: 10.1038/s41419-024-06882-5

Figure Lengend Snippet: A , B The viability of ES cells, specifically the A673 and RDES cell lines, was assessed following treatment with the top 10 anticancer compounds. C Bone marrow stem cells (BMSC) served as a normal control group. Both ES and normal BMSC cells were exposed to UNC0379 at the indicated concentration for 48 h, after which cell viability was measured. D The colony formation assay demonstrated a significant reduction in the ability of A673, RDES, and SKNMC cells to form colonies after treatment with UNC0379 at 2 μM, as compared to the DMSO control group. E The impact of SETD8 knockdown using short hairpin RNA (shRNA) on cell proliferation was evaluated through a colony formation assay. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The RDES and SKNMC human ES cell lines, along with BMSC, were procured from Jennio Biotech Co., Ltd. Additionally, the A673 and SK-NEP-1 human ES cell lines were sourced from Procell, Wuhan, China.

Techniques: Control, Concentration Assay, Colony Assay, Knockdown, shRNA

Journal: Cell Death & Disease

Article Title: SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

doi: 10.1038/s41419-024-06882-5

Figure Lengend Snippet: A WB analysis was conducted to assess the expression levels of the SETD8 protein in BMSC and across four ES cell lines. The original blots corresponding to these experiments are displayed in Fig. . B IHC provided representative images of SETD8 protein expression in ES patient samples at a magnification of ×200. C , D Kaplan–Meier plots depicted the overall survival (OS) and event-free survival (EFS) for ES patients with high versus low SETD8 expression levels. E The impact of UNC0379 (2 μM) on the clonogenic capacity of ES cells, in the context of various cell death inhibitors (10 μM Z-VAD-FMK, necrostatin-1, Ferrostatin-1, deferoxamine, 5 mM 3-Methyladenine and 200 μM Methyladenine), was evaluated through a colony formation assay. F The viability of A673 and RDES cells following 48 h of exposure to UNC0379 (2 μM) with or without the aforementioned cell death inhibitors was measured using the CCK-8 assay. G A column chart illustrated the variation in apoptosis rates among the ES cell lines following SETD8 inhibition by UNC0379. H The relative levels of intracellular iron (Fe2+) in ES cells were measured after SETD8 was inhibited by UNC0379. I The levels of reactive oxygen species (ROS) within ES cells were determined post-SETD8 inhibition by UNC0379. J The lipid ROS levels in ES cells were assessed after SETD8 inhibition by UNC0379. Data are presented as the mean ± standard deviation from three separate experiments. ‘ns’ denotes no statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The RDES and SKNMC human ES cell lines, along with BMSC, were procured from Jennio Biotech Co., Ltd. Additionally, the A673 and SK-NEP-1 human ES cell lines were sourced from Procell, Wuhan, China.

Techniques: Expressing, Colony Assay, CCK-8 Assay, Inhibition, Standard Deviation

Journal: Cell Death & Disease

Article Title: SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

doi: 10.1038/s41419-024-06882-5

Figure Lengend Snippet: A The clonogenic potential of SETD8 knockdown in ES cells was evaluated in the presence or absence of specific cell death inhibitors, including 10 μM Z-VAD-FMK, and 10 μM Ferrostatin-1, using a colony formation assay. B A673 and RDES cells underwent SETD8 knockdown and were treated with the aforementioned inhibitors for 48 h, after which their viability was measured using the CCK-8 assay. C A column chart displayed the shifts in apoptosis rates among the ES cell lines subsequent to SETD8 knockdown. D The levels of intracellular ROS in ES cells were quantified following SETD8 knockdown. E Intracellular lipid ROS levels were similarly measured in ES cells post-SETD8 knockdown. F Relative changes in intracellular Fe2+ levels were observed in ES cells after SETD8 knockdown. The data are expressed as the mean ± standard deviation derived from three individual experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The RDES and SKNMC human ES cell lines, along with BMSC, were procured from Jennio Biotech Co., Ltd. Additionally, the A673 and SK-NEP-1 human ES cell lines were sourced from Procell, Wuhan, China.

Techniques: Knockdown, Colony Assay, CCK-8 Assay, Standard Deviation, Derivative Assay

Journal: Cell Death & Disease

Article Title: SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

doi: 10.1038/s41419-024-06882-5

Figure Lengend Snippet: A , B The KEGG pathway analysis enriched by DEGs from A673 and RDES after SETD8 suppression by siRNA. C , D mRNA expression levels of DEGs in the MAPK pathway validated in ES cells after SETD8 genetic knockdown by siRNA. E Results of WB analysis to confirm the protein expression changes of RAC3, ERK1/2, and p-ERK1/2 in the MAPK pathway after SETD8 inhibition. The original blots corresponding to these experiments are displayed in Fig. . F Results of WB analysis to confirm the protein expression changes of ERK1/2 and p-ERK1/2 after RAC3 knockdown. The original blots corresponding to these experiments are displayed in Fig. . G Colony formation assay after RAC3 knockdown in ES cells. H Colony formation assay determined the reproductive ability of ES cells with RAC3 knockdown in the absence or presence of indicated cell death inhibitors (10 µM Z-VAD-FMK and 10 µM Ferrostatin-1). I A673 and RDES cells with RAC3 knockdown in the absence or presence of indicated cell death inhibitors (10 µM Z-VAD-FMK and 10 µM Ferrostatin-1) for 48 h, cell viability was assayed with CCK-8. J Changes of apoptosis rates after RAC3 inhibition in ES cells. K Changes of the intracellular ROS levels after RAC3 inhibition in ES cells. L Changes of the intracellular lipid ROS levels after RAC3 inhibition in ES cells. M Changes of the relative intracellular Fe2+ levels in ES cells after RAC3 inhibition. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The RDES and SKNMC human ES cell lines, along with BMSC, were procured from Jennio Biotech Co., Ltd. Additionally, the A673 and SK-NEP-1 human ES cell lines were sourced from Procell, Wuhan, China.

Techniques: Expressing, Knockdown, Inhibition, Colony Assay, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

doi: 10.1038/s41419-024-06882-5

Figure Lengend Snippet: A Silver staining demonstrated co-immunoprecipitation using an antibody against SETD8 in A673 cells. B Mass spectrometry data confirmed the physical association between SETD8 and YBX1 proteins. C Immunoprecipitation results validated the binding interaction between SETD8 and YBX1 in ES cells. The original blots corresponding to these experiments are displayed in Fig. . D WB analysis detected changes in the expression levels of phosphorylated YBX1 at serine102 (p-Ser102 YBX1) and total YBX1 in ES cell lines following SETD8 knockdown. The original blots corresponding to these experiments are displayed in Fig. . E Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed alterations in YBX1 mRNA levels in ES cell lines post-SETD8 knockdown. F The clonogenic capacity of ES cells post YBX1 knockdown was illustrated through a colony formation assay. G The reproductive potential of YBX1 knockdown in ES cells was evaluated in the context of specific cell death inhibitors (10 μM Z-VAD-FMK and Ferrostatin-1) using a colony formation assay. H A column chart, based on CCK-8 assay results, depicted cell viability following YBX1 knockdown with or without the specified cell death inhibitors for 48 h. I A column chart showed the shifts in apoptosis rates across ES cell lines after YBX1 was suppressed using siRNA. J Relative changes in intracellular Fe2+ levels were observed in ES cells after YBX1 inhibition. K Intracellular ROS levels were measured in ES cells following YBX1 inhibition. L Intracellular lipid ROS levels were assessed in ES cells after YBX1 inhibition. Data are presented as mean ± standard deviation from three separate experiments. ‘ns’ signifies no statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The RDES and SKNMC human ES cell lines, along with BMSC, were procured from Jennio Biotech Co., Ltd. Additionally, the A673 and SK-NEP-1 human ES cell lines were sourced from Procell, Wuhan, China.

Techniques: Silver Staining, Immunoprecipitation, Mass Spectrometry, Binding Assay, Expressing, Knockdown, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Colony Assay, CCK-8 Assay, Inhibition, Standard Deviation

Journal: Cell Death & Disease

Article Title: SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

doi: 10.1038/s41419-024-06882-5

Figure Lengend Snippet: A qRT-PCR analysis verified the expression of RAC3 mRNA subsequent to the inhibition of YBX1. B ChIP-PCR confirmed the presence of YBX1 bound to the RAC3 promoter region in ES cells. C ChIP-qPCR was utilized to assess the alterations in YBX1 binding at the RAC3 promoter in A673 and RDES cells with or without SETD8 knockdown. D , E WB analysis determined the distribution of YBX1 in the cytoplasmic and nuclear fractions after SETD8 knockdown for a duration of 48 h. LaminB1 served as a nuclear fraction loading control, while GAPDH was used as a cytoplasmic fraction loading control. The original blots corresponding to these experiments are displayed in Fig. . F Immunofluorescence (IF) assays revealed the cellular localization of the YBX1 protein in ES cells following SETD8 knockdown for 48 h. Data are presented as mean ± standard deviation from three separate experiments. * p < 0.05, ** p < 0.01.

Article Snippet: The RDES and SKNMC human ES cell lines, along with BMSC, were procured from Jennio Biotech Co., Ltd. Additionally, the A673 and SK-NEP-1 human ES cell lines were sourced from Procell, Wuhan, China.

Techniques: Quantitative RT-PCR, Expressing, Inhibition, Binding Assay, Knockdown, Control, Immunofluorescence, Standard Deviation