Journal: Viruses

Article Title: Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

doi: 10.3390/v15020363

Figure Lengend Snippet: Virotherapeutic treatment of GFP/luc-labeled human HT-29 tumor cells in cell culture with GLV-0b347. ( A ) Schematic illustration of the three-step virotherapeutic process and associated detection capabilities: (1) GFP/luc-labeled HT-29 tumor cells were seeded into a 24-well cell culture plate. Successful plating of the cells can be verified by the determination of GFP fluorescence. (2) The treatment of HT-29 cells with oncolytic viruses encoding a red-fluorescent marker protein. The successful infection of the tumor cells can be verified by the determination of red fluorescence. (3)/(4) The viral oncolysis can be determined by a decrease in GFP as well as in luciferase activity. Over time, enhanced red fluorescence indicates an increasing number of tumor cells being infected by the red-fluorescence marker gene encoding virotherapeutic compounds. ( B ) The fluorescence images of HT-29 GFP/luc-labeled cells at 72 h postinfection (hpi) with GLV-0b347 at different multiplicities of infection (MOIs), as depicted. BF, brightfield; OL, overlay of GFP and TurboFP635 signal.

Article Snippet: The human GFP/Luciferase (luc) dual-labeled HT-29 cancer cell line was purchased from GeneCopoeia TM (Cat. SCL-C06-HLG, Rockville, MD, USA) and authenticated by short tandem repeat analysis (Eurofins, Ebersberg, Germany).

Techniques: Labeling, Cell Culture, Fluorescence, Marker, Infection, Luciferase, Activity Assay

Journal: Viruses

Article Title: Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

doi: 10.3390/v15020363

Figure Lengend Snippet: Virotherapeutic treatment of GFP/luc-labeled human HT-29 tumor cells in cell culture with MeV-DsRed. Fluorescence images of HT-29 GFP/luc-labeled cells at 72 h postinfection (hpi) with MeV-DsRed at different multiplicities of infection (MOIs), as depicted. BF, brightfield; OL, overlay of GFP and DsRed signal.

Article Snippet: The human GFP/Luciferase (luc) dual-labeled HT-29 cancer cell line was purchased from GeneCopoeia TM (Cat. SCL-C06-HLG, Rockville, MD, USA) and authenticated by short tandem repeat analysis (Eurofins, Ebersberg, Germany).

Techniques: Labeling, Cell Culture, Fluorescence, Infection

Journal: Viruses

Article Title: Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

doi: 10.3390/v15020363

Figure Lengend Snippet: Comparison of different detection options for the oncolytic activity of virotherapeutic compounds GLV-0b347 ( A , images to the left) and MeV-DsRed ( B , images to the right) in HT-29 GFP/luc tumor cells. HT-29 GFP/luc cells were infected with GLV-0b347 ( A ) or MeV-DsRed ( B ) at different multiplicities of infection (MOIs) ranging from 0.0001 to 1 for GLV-0b347, from 0.001 to 10 for MeV-DsRed, or remained uninfected (MOCK). At 72 h postinfection (hpi), remaining tumor cell masses were determined by either (i) SRB viability assays, (ii) the measurement of the luciferase activity, or (iii) the quantification of the GFP or red-fluorescence intensity. Each measurement was calculated relative to the MOCK control. The mean ± SD of at least two independent experiments performed in triplicate is shown. ANOVA test relative to MOCK-infected control: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The human GFP/Luciferase (luc) dual-labeled HT-29 cancer cell line was purchased from GeneCopoeia TM (Cat. SCL-C06-HLG, Rockville, MD, USA) and authenticated by short tandem repeat analysis (Eurofins, Ebersberg, Germany).

Techniques: Activity Assay, Infection, Luciferase, Fluorescence

Journal: Viruses

Article Title: Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

doi: 10.3390/v15020363

Figure Lengend Snippet: Human ex vivo peritoneum model and schematic illustration of the three-step virotherapeutic process in co-cultures with GFP/luc-labeled human HT-29 tumor cells. ( A ) Photographic image of the human ex vivo peritoneal model cultivated between stainless steel rings in a 24-well plate. ( B ) Photographic image of the peritoneum in the ex vivo model through a light microscope. ( C ) (1) Preparation of co-cultures of the peritoneum from noncancer patients and human GFP/luc-labeled HT-29 tumor cells. Successful plating of the cells can be verified by fluorescence microscopy. (2) Virotherapeutic treatment of co-cultures with oncolytic viruses carrying a red-fluorescent marker protein. Successful infection of the tumor cells can be verified by the determination of red fluorescence via fluorescence microscopy. (3) Viral oncolysis can be determined by a decrease in GFP and red fluorescence as well as by a decrease in luciferase activity.

Article Snippet: The human GFP/Luciferase (luc) dual-labeled HT-29 cancer cell line was purchased from GeneCopoeia TM (Cat. SCL-C06-HLG, Rockville, MD, USA) and authenticated by short tandem repeat analysis (Eurofins, Ebersberg, Germany).

Techniques: Ex Vivo, Labeling, Light Microscopy, Fluorescence, Microscopy, Marker, Infection, Luciferase, Activity Assay

Journal: Viruses

Article Title: Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

doi: 10.3390/v15020363

Figure Lengend Snippet: Virotherapeutic treatment of PC models with recombinant vaccinia virus GLV-0b347. The fluorescence images of GLV-0b347-infected co-cultures (( A ); 1.5 × 10 6 plaque-forming units (PFU)) or MOCK-infected ( B ) co-cultures consisting of the peritoneum of noncancer patients and adherently growing GFP/luc-labeled human HT-29 tumor cells at days 2, 4, and 7 postinfection (dpi); original magnification 4×. ( C ) Luciferase activity of GFP/luc–HT-29 cells growing on peritoneum at 2, 4, and 7 dpi, either GLV-0b347-infected or MOCK-infected. Each measurement is normalized to the MOCK control. The mean ± SD of one experiment performed in triplicates is shown. t -test relative to MOCK-infected control: * p < 0.05 and ** p < 0.01. ns; not significant. ( D ) The hematoxylin and eosin staining of human peritoneal tissue with and w/o co-culture of GFP/luc-labeled HT-29 cells at 7 dpi, either GLV-0b347-infected or MOCK-infected. ( E ) The EpCAM staining of peritoneal tissue with the co-culture of HT-29 cells at 7 dpi, either GLV-0b347-infected or MOCK-infected. ( F ) The vaccinia virus staining of peritoneal tissue with co-culture of HT-29 cells at 7 dpi, either GLV-0b347-infected or MOCK-infected. Experiments were conducted with the peritoneal tissue from different patients and show representative data from at least three different experiments. P; peritoneum. Black arrows indicate intact or infected and lysed HT-29 cells on the surface of the peritoneum. Scale bars represent 100 μm.

Article Snippet: The human GFP/Luciferase (luc) dual-labeled HT-29 cancer cell line was purchased from GeneCopoeia TM (Cat. SCL-C06-HLG, Rockville, MD, USA) and authenticated by short tandem repeat analysis (Eurofins, Ebersberg, Germany).

Techniques: Recombinant, Fluorescence, Infection, Labeling, Luciferase, Activity Assay, Staining, Co-Culture Assay

Journal: Viruses

Article Title: Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

doi: 10.3390/v15020363

Figure Lengend Snippet: Virotherapeutic treatment of PC models with recombinant measles vaccine virus MeV-DsRed. The fluorescence images of MeV-DsRed-infected co-cultures (( A ); 1.5 × 10 6 plaque-forming units (PFU)) or MOCK-infected ( B ) co-cultures consisting of the peritoneum of noncancer patients and GFP/luc-labeled human HT-29 tumor cells at days 2, 4, and 7 postinfection (dpi); original magnification 4×. ( C ) The luciferase activity of GFP/luc–HT-29 cells growing on peritoneum at 2, 4, and 7 dpi, either MeV-DsRed-infected or MOCK-infected. Each measurement is normalized to the MOCK control. The mean ± SD of one experiment performed in triplicates is shown. t -test relative to MOCK-infected control: * p < 0.05. ns; not significant. ( D ) The hematoxylin and eosin staining of human peritoneal tissue with and w/o co-culture of GFP/luc-labeled HT-29 tumor cells at 7 dpi with MeV-DsRed or MOCK infection. ( E ) The EpCAM staining of peritoneal tissue with co-culture of HT-29 cells at 7 dpi with MeV-DsRed or MOCK infection. ( F ) The MeV staining of peritoneal tissue with co-culture of HT-29 cells at 7 dpi with MeV-DsRed or MOCK infection. The experiments were conducted with peritoneal tissue from different patients and show representative data from at least three different experiments. P; peritoneum. Black arrows indicate intact or infected and lysed HT-29 tumor cells on the surface of the peritoneum. Scale bars represent 100 μm.

Article Snippet: The human GFP/Luciferase (luc) dual-labeled HT-29 cancer cell line was purchased from GeneCopoeia TM (Cat. SCL-C06-HLG, Rockville, MD, USA) and authenticated by short tandem repeat analysis (Eurofins, Ebersberg, Germany).

Techniques: Recombinant, Fluorescence, Infection, Labeling, Luciferase, Activity Assay, Staining, Co-Culture Assay

Journal: Archives of Toxicology

Article Title: The safety of nanostructured synthetic amorphous silica (SAS) as a food additive (E 551)

doi: 10.1007/s00204-016-1850-4

Figure Lengend Snippet: Genotoxicity of silica in vitro (including data of non-food-grade and colloidal SAS)

Article Snippet: HT-29 human colon carcinoma cell line , Pyrogenic SAS (AEROSIL ® 200, AEROSIL ® Ox50) , 12, 40 nm, 200, 50 m 2 /g , Evonik Industries , Cytotoxicity (±FCS, 1 and 10 %, 0.03–156.3 μg/cm 2 ); comet assay with and without Fpg , Negative, no oxidative DNA damage , Gehrke et al. ( ) .

Techniques: In Vitro, Mutagenesis, Spot Test, Derivative Assay, Single Cell Gel Electrophoresis, Incubation, Positive Control, Alkaline Single Cell Gel Electrophoresis

Journal: Molecular Therapy. Nucleic Acids

Article Title: Antitumor effects of chemically modified miR-143 lipoplexes in a mouse model of pelvic colorectal cancer via myristoylated alanine-rich C kinase substrate downregulation

doi: 10.1016/j.omtn.2023.102079

Figure Lengend Snippet: Features of CRC pelvic recurrence mouse model (A) Schema of our mouse model. The needle was navigated toward the pelvic cavity ∼1.0 cm from the skin, and CRC cells (DLD-1 clone#1-Luc and HT-29-Luc) were injected. The tumor grew gradually in the pelvic cavity, compressing the rectum, as the yellow arrows indicate. (B) The black point represents the injection site in the centroid of the triangle defined by the vagina, anus, and ischial spine. (C) Representative anatomical image of the mouse model. The pelvic CRC tumor is circled in blue. Pressure from the tumor caused the distension of the rectum (white arrowhead). (D) Representative IVIS images show an overall view of the mouse. The white arrowhead indicates the pelvic CRC tumor. The scale bar on the right indicates the strength of fluorescence. (E) Representative scout view. CT imaging was conducted within the area indicated by the purple square of the scout view. The yellow, blue, and red squares indicated the inferior, middle, and superior regions, respectively, of the pelvis. (F) Representative CT of the orange pelvic region. The tumor is marked by the orange arrow, and the orange arrowhead marks the rectum. The tumor did not press on the rectum in this area (scale bar: 5 mm) (G) Representative CT image of the blue pelvic region (scale bar: 5 mm). The connective tissue surrounding the CRC tumor appeared white in the CT imaging, indicating that the connective tissue was rich in blood flow (blue arrow). The rectum was deformed due to compression by the tumor (blue arrowhead). (H) Representative CT image of the red pelvic region (scale bar: 5 mm). The red arrow marks the tumor with rich blood flow in connective tissue. The red arrowhead indicates that the rectum was distended, with notable stool retention within the rectal lumen.

Article Snippet: Human CRC cell lines (DLD-1, RRID: CVCL 0248 and HT-29, RRID: CVCL 0320) were obtained from Taiho Pharmaceutical (Shibuya-ku, Tokyo, Japan).

Techniques: Injection, Fluorescence, Imaging

Journal: Molecular Therapy. Nucleic Acids

Article Title: Antitumor effects of chemically modified miR-143 lipoplexes in a mouse model of pelvic colorectal cancer via myristoylated alanine-rich C kinase substrate downregulation

doi: 10.1016/j.omtn.2023.102079

Figure Lengend Snippet: Stability of CM-miR-143 lipoplexes in mouse blood and delivery to pelvic CRC tumors (A) Scheme of in vitro experiments for measuring miR-143 levels using qRT-PCR. Naked miRNA was incubated in FBS. Retention rates of miR-143 were measured at 0, 10, 30, and 60 min after incubation. (B) Blue, purple, and orange lines indicate control (C) miRNA, Am-miR-143 (Am), and CM-miR-143 (CM), respectively. Values at 0 min for Am-miR-143 and CM-miR-143 are indicated as 1.0. After incubation for 10 min, Am-miR-143 levels decreased significantly. CM-miR-143 levels remained high until 60 min after incubation. Data are presented as mean ± SEM (∗p < 0.05; ∗∗∗p < 0.001; n = 3). (C) Scheme of in vivo experiments for measuring plasma miR-143 levels using qRT-PCR. Each miRNA lipoplex (330 μg/kg miRNA per administration) was injected into the tail vein once. Blood miR-143 levels were measured at 12, 24, 48, and 72 h after administration. (D) Purple and orange lines indicate miR-143 ratios in the Am-miR-143 and CM-miR-143 groups, respectively. miR-143 levels at 12 h are indicated as 1.0. At 24 h after administration, Am-miR-143 levels decreased, whereas CM-miR-143 levels remained high until 72 h. Data are presented as the mean ± SEM (∗p < 0.05; ∗∗p < 0.01; n = 7). (E) Scheme of in vivo experiments for in situ hybridization and qRT-PCR, aiming to characterize miR-143-143 distribution in pelvic CRC tumors (DLD-1 clone#1-Luc cells). In situ hybridization was performed using tissue samples from pelvic CRC tumors resected 10 min after miRNA lipoplex administration; qRT-PCR was performed 24 and 72 h after administration (330 μg/kg miRNA per administration). (F) Representative images from in situ hybridization and CT. Purple signals in situ hybridization indicate miR-143. The images illustrate 3 regions: the border area, tumor, and connective tissue in situ hybridization. The border area represents the region surrounding the tumor. The connective tissue, highlighted by an orange square in CT imaging, is a magnified image of the border area. CT imaging showed that blood flow was rich in connective tissue (scale bar: 5 mm). (G) Results from qRT-PCR revealed that miR-143 levels were significantly higher at 24 h in the CM-miR-143 group than in the control group. However, the 2 groups did not differ significantly in miR-143 levels by 72 h. Data are presented as mean ± SEM (∗p < 0.05; n = 4). ns, not significant; Adm, administration.

Article Snippet: Human CRC cell lines (DLD-1, RRID: CVCL 0248 and HT-29, RRID: CVCL 0320) were obtained from Taiho Pharmaceutical (Shibuya-ku, Tokyo, Japan).

Techniques: In Vitro, Quantitative RT-PCR, Incubation, Control, In Vivo, Clinical Proteomics, Injection, In Situ Hybridization, Imaging

Journal: Molecular Therapy. Nucleic Acids

Article Title: Antitumor effects of chemically modified miR-143 lipoplexes in a mouse model of pelvic colorectal cancer via myristoylated alanine-rich C kinase substrate downregulation

doi: 10.1016/j.omtn.2023.102079

Figure Lengend Snippet: Systemic administration of CM-miR-143 lipoplexes suppressed tumor growth of CRC pelvic recurrence mouse (A) Scheme of in vivo experiments to measure fluorescence. Mice were randomly divided into 2 groups after tumor formation. To measure fluorescence, miRNA lipoplexes (330 μg/kg miRNA per administration) were injected into the tail vein 3 times over 10 days. (B) All IVIS images of control (C) and CM-miR-143 (CM-143) at days 0, 3, 7, and 10 posttreatment in the DLD-1 clone#1-Luc mouse model and the HT-29-Luc mouse model are shown. (C) Fluorescence intensity was measured on days 0, 3, 7, and 10 posttreatment using IVIS in DLD-1 clone#1-Luc and HT-29-Luc mouse models (DLD-1 clone#1-Luc: n = 11, HT-29-Luc: n = 11). The graph shows variation in fluorescence intensity over time. Fold changes in fluorescence intensity were analyzed per group. In the DLD-1 clone#1-Luc mouse model, the CM-miR-143 group experienced significantly suppressed fluorescence intensity on days 3, 7, and 10 posttreatment compared with the control group; in the HT-29-Luc mouse model, significant suppression occurred only on day 10. Black arrowheads indicate the injection of miRNA lipoplexes into the tail vein. Data are presented as mean ± SEM (∗p < 0.05).

Article Snippet: Human CRC cell lines (DLD-1, RRID: CVCL 0248 and HT-29, RRID: CVCL 0320) were obtained from Taiho Pharmaceutical (Shibuya-ku, Tokyo, Japan).

Techniques: In Vivo, Fluorescence, Injection, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Antitumor effects of chemically modified miR-143 lipoplexes in a mouse model of pelvic colorectal cancer via myristoylated alanine-rich C kinase substrate downregulation

doi: 10.1016/j.omtn.2023.102079

Figure Lengend Snippet: Systemic administration of CM-miR-143 lipoplexes prolonged CRC pelvic recurrence mouse survival (A) All IVIS images of control and CM-miR-143 before treatment in the DLD-1 clone#1-Luc and HT-29-Luc mouse models. (B) The relative fluorescence intensity of control (C) and CM-miR-143 (CM-143) before treatment in the DLD-1 clone#1-Luc and HT-29-Luc mouse models. There was no significant difference between the 2 groups. (n = 15) (C) The body weight of control and CM-miR-143 before treatment of the DLD-1 clone#1-Luc and HT-29-Luc mouse models. There was no significant difference in body weight between the 2 groups (n = 15). (D) Kaplan-Meier plots show the survival rate in control (blue line) and CM-miR-143 (orange line) groups in the DLD-1 clone#1-Luc and HT-29-Luc mouse models (n = 15). The significance of survival duration was determined using log rank survival analysis. (E) Body weights did not differ significantly between control and CM-miR-143 groups in either the DLD-1 clone#1-Luc or the HT-29-Luc mouse models after treatment (n = 15). ns, not significant. Data are presented as the mean ± SEM.

Article Snippet: Human CRC cell lines (DLD-1, RRID: CVCL 0248 and HT-29, RRID: CVCL 0320) were obtained from Taiho Pharmaceutical (Shibuya-ku, Tokyo, Japan).

Techniques: Control, Fluorescence

Journal: Molecular Therapy. Nucleic Acids

Article Title: Antitumor effects of chemically modified miR-143 lipoplexes in a mouse model of pelvic colorectal cancer via myristoylated alanine-rich C kinase substrate downregulation

doi: 10.1016/j.omtn.2023.102079

Figure Lengend Snippet: CM-miR-143 lipoplexes downregulated MARCKS expression in vivo (A) Scheme of in vivo experiment for proteome, IHC, and western blot analyses. In the DLD-1 clone#1-Luc model, miRNA lipoplexes (330 μg/kg miRNA per administration) were injected into the tail vein 4 times in 2 weeks before tissue samples were obtained from pelvic CRC tumors for the 3 analyses. In the HT-29-Luc model, miRNA lipoplexes (330 μg/kg miRNA) were administered twice in 1 week before tissue samples from pelvic CRC tumors were obtained for western blotting. (B) Heatmap showing results of 4,158 proteins from proteome analysis of DLD-1 clone#1-Luc tumor samples. Heatmap showing the results of 3 samples in the control and CM-miR-143 groups. The purple intensity indicates the expression of the protein. The right-side panel indicates that the expression of MARCKS is downregulated. (C) Volcano plot showing results from proteome analysis of control and CM-miR143 groups (n = 3). The red point indicates MARCKS expression. Negative log2 fold change: higher expression in the control group and lower expression in the CM-miR143 group. Positive log2 fold change: lower expression in the control group and higher expression in the CM-miR143 group. (D) Representative IHC images for MARCKS in control and CM-miR-143 groups. MARCKS downregulation was observed in the CM-miR-143 group. (E) Western blotting to evaluate p-MARCKS, MARCKS, p-AKT, and AKT expression. p-MARCKS and MARCKS protein expression was significantly downregulated in the CM-miR-143 group. p-AKT and AKT levels did not differ between the control and CM-miR-143 groups. β-Actin was used as an internal control. (F) p-MARCKS and MARCKS band intensities in the western blots were calculated using ImageJ. MARCKS and p-MARCKS expression was significantly downregulated in pelvic CRC tumors in the DLD-1 clone#1-Luc and HT-29-Luc models. Data are presented as mean ± SEM (∗p < 0.05; n = 4).

Article Snippet: Human CRC cell lines (DLD-1, RRID: CVCL 0248 and HT-29, RRID: CVCL 0320) were obtained from Taiho Pharmaceutical (Shibuya-ku, Tokyo, Japan).

Techniques: Expressing, In Vivo, Western Blot, Injection, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Antitumor effects of chemically modified miR-143 lipoplexes in a mouse model of pelvic colorectal cancer via myristoylated alanine-rich C kinase substrate downregulation

doi: 10.1016/j.omtn.2023.102079

Figure Lengend Snippet: CM-miR-143 inhibited CRC cell proliferation by downregulating MARCKS in vitro (A) Schematic of putative miR-143 seed sequence binding at the MARCKS 3ʹ UTR region (WT) and MUT sequence of the putative binding site. Red letters are binding sites and mutation sequences used for luciferase assays in MUT. (B) Am-miR-143 significantly inhibited luciferase activity in WT. Activities were completely recovered to the control levels in MUT. Data are presented as mean ± SEM (∗p < 0.05; n = 6). ns, not significant. (C) Cell viability at 72 h after transfection of each miRNA (10 nM) with lipofectamine RNAiMAX in the DLD-1 or HT-29 cells. CM-miR-143 significantly suppressed cell viability in both cell lines compared with control miRNA or Am-miR-143. Data are presented as mean ± SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 3). (D) Western blotting to evaluate p-MARCKS, MARCKS, p-AKT, and AKT protein expression at 72 h posttransfection in DLD-1 or HT-29 cells. All of the proteins were significantly downregulated in the CM-miR-143 group. β-Actin was used as an internal control. (E) Cell viability at 72 h after siRNA-MARCKS transfection (5 nM) in DLD-1 or HT-29 cells. Compared with control siRNA, siRNA-MARCKS 1 and 2 significantly suppressed cell growth in both cell lines. Data are presented as mean ± SEM (∗∗∗p < 0.001; n = 3). (F) Western blotting to evaluate p-MARCKS, MARCKS, p-AKT, and AKT protein expression at 72 h after transfection in DLD-1 or HT-29 cells. p-MARCKS and MARCKS were downregulated after transfection of siRNA-MARCKS 1 or 2, whereas p-AKT and AKT were not downregulated. β-Actin was used as an internal control. si-MARCKS1, si1; si-MARCKS2, si2.

Article Snippet: Human CRC cell lines (DLD-1, RRID: CVCL 0248 and HT-29, RRID: CVCL 0320) were obtained from Taiho Pharmaceutical (Shibuya-ku, Tokyo, Japan).

Techniques: In Vitro, Sequencing, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Control, Transfection, Western Blot, Expressing