Journal: Molecular cancer research : MCR

Article Title: A Functional Precision Oncology Approach to Identify Treatment Strategies for Myxofibrosarcoma Patients

doi: 10.1158/1541-7786.MCR-21-0255

Figure Lengend Snippet: (A, B) Ex vivo drug testing of the novel patient derived cell model (WCM197) together with the OH931, NMFH-1 and HFF (benign human fibroblasts) using abemaciclib a second line CDK4/6-inhibitor and pralatrexate an antifolate inhibitor in a 6–8-point dilution dose response log scale to determine the IC50 values, highest dose was 10uM and lowest dose was 4.7 pM with an assay time of 96 hours. Greater cytostatic and cytotoxic effect was seen in the cells with the CDKN2A/B and MTAP null status (WCM197 and OH931) compared to CDKN2A/B and MTAP wild type status (NMFH-1 and HFF). (C) The combination of pralatrexate (IC30) and abemaciclib killed all our patient cells within 48 hours compared to the vehicle and abemaciclib and pralatrexate mono-therapy. (D) WCM197 sarco-sphere formation is completely inhibited with the combination of abemaciclib and pralatrexate. (E) Drug combination of abemaciclib and pralatrexate causes massive cell swellings and cytoplasmatic vacuole formation. (F) Cleaved caspase 3 and cleaved PARP at 24, 48 and 72 hours were detected with the combination of abemaciclib and pralatrexate indicating the induction of cell death in the tumor cells after treatment.

Article Snippet: The Human Foreskin Fibroblast (HFF) cells were provided by Dr. Carla Grandori, SEngine Precision Medicine, Seattle, WA, USA as a control specimen for high throughput drug screening ( 22 ).

Techniques: Ex Vivo, Derivative Assay

Journal: Molecular cancer research : MCR

Article Title: A Functional Precision Oncology Approach to Identify Treatment Strategies for Myxofibrosarcoma Patients

doi: 10.1158/1541-7786.MCR-21-0255

Figure Lengend Snippet: (A) High throughput drug screening of all 4 cell strains (HFF, OH931, WCM197, NMFH-1) shown as a heat map (blue is sensitive to red resistant). RB1 wt and CDKN2A/B/MTAP null cells (WCM197 and OH931) show higher sensitivity to CDK4/6 inhibititors such as abemaciclib (indicated by the yellow arrows) and the antifolate pralatrexate and methotrexate (indicated by the green arrows) compared to the CDKN2A/B/MTAP wild type lines (NMFH-1 and HFF). Selective sensitivity to PLK-1 inhibitors was detected in WCM197 and OH931 cells compared to the NMFH-1 and the control line HFF, shown in orange arrows. (B) Graphs show the response of the sarcoma cells to each compound in the library as area under the curve (AUC) compared to the benign human foreskin fibroblast line (HFF) as a normal control. (C-D) Ex vivo drug validation of the two PLK-1 inhibitors (volasertib and rigosertib) confirms high sensitivity for WCM197 and OH931 cells. (E, F) WCM197 3D sarco-spheres are completely inhibited in growth under volasertib treatment and show cell death indicated by cleaved caspase 3, PARP and cleaved PARP. Cell death was monitored with cleaved caspase 3 and cleaved PARP over 96 hours with a peak at 24–72 hours.

Article Snippet: The Human Foreskin Fibroblast (HFF) cells were provided by Dr. Carla Grandori, SEngine Precision Medicine, Seattle, WA, USA as a control specimen for high throughput drug screening ( 22 ).

Techniques: High Throughput Screening Assay, Drug discovery, Control, Ex Vivo, Biomarker Discovery

Journal: mBio

Article Title: Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile

doi: 10.1128/mBio.01237-16

Figure Lengend Snippet: Culture supernatant fluids collected from the agrB1D1 mutants are not cytotoxic to human foreskin fibroblast cells. Strain 630 and R20291 agrB1D1 and agrB2D2 mutants were cultured for 48 h in BHI medium anaerobically at 37°C. The culture supernatant fluids were collected, filter sterilized with a 0.45-µm filter, and examined for cytotoxicity with the Bartels Clostridium difficile cytotoxicity assay kit (Trinity Biotech, Jamestown, NY). The culture fluids were incubated with the fibroblast cells for 24 h and observed under a microscope for cytotoxic effects. Images were taken with an EVOS XL microscope (Advanced Microscopy Group) at ×20 magnification. Panels: A, a representative image of fibroblast cells cultured in growth medium only; B, wild-type 630; C, 630 agrB1D1 mutant; D, 630 complemented agrB1D1 mutant; E, wild-type R20291; F, R20291 agrB1D1 mutant; G, complemented R20291 agrB1D1 mutant; H, R20291 agrB2D2 mutant.

Article Snippet: Ready-made human foreskin fibroblast cells (Trinity Biotech, Jamestown, NY) in 150 μl of medium were treated with 50 μl of culture supernatant fluids from the wild-type and agr mutant strains and incubated for 48 h at 37°C.

Techniques: Cell Culture, Cytotoxicity Assay, Incubation, Microscopy, Mutagenesis

Journal: mBio

Article Title: Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile

doi: 10.1128/mBio.01237-16

Figure Lengend Snippet: Culture supernatants from C. difficile agrB1D1 mutants have no effect on cell viability. Strain 630 (A) and R20291 (B) agrB1D1 and agrB2D2 mutants were cultured for 48 h in BHI medium anaerobically at 37°C. The culture supernatant fluids were collected, filter sterilized with a 0.45-µm filter, and tested for their effect on cell viability. Briefly, ready-made fibroblast cells (Trinity Biotech, Jamestown, NY) were treated with the culture supernatant fluids and incubated for 48 h at 37°C aerobically. Following the incubation period, cell viability was examined with the CellTiter 96 nonradioactive cell proliferation assay (Promega, Madison, WI) in accordance with the instructions provided by the manufacturer. agrB1D1 Mut, agrB1D1 deletion mutant; agrB1D1 Mut+Comp, agrB1D1 mutant complemented with a plasmid bearing the wild-type agrB1D1 locus; agrB2D2 Mut, R20291 agrB2D2 deletion mutant. The differences between the amounts of formazan produced by fibroblast cells treated with supernatants from the agrB1D1 mutants and wild-type strains, the complemented agrB1D1 mutants, and the R20291 agrB2D2 mutant were significant ( P = 0.0012). Error bars represent the standard deviations of three independent experiments.

Article Snippet: Ready-made human foreskin fibroblast cells (Trinity Biotech, Jamestown, NY) in 150 μl of medium were treated with 50 μl of culture supernatant fluids from the wild-type and agr mutant strains and incubated for 48 h at 37°C.

Techniques: Cell Culture, Incubation, Proliferation Assay, Mutagenesis, Plasmid Preparation, Produced