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: Molecular Therapy Oncology

Article Title: Neospora caninum as delivery vehicle for anti-PD-L1 scFv-Fc: A novel approach for cancer immunotherapy

doi: 10.1016/j.omton.2025.200968

Figure Lengend Snippet: Generation of a recombinant N. caninum secreting scFv-Fc (A) Schematic representation of pUC5-scFv-Fc vector used for the obtention of recombinant Nc-1-scFv-Fc strain. Genetic map of pUC5 plasmid including the sequence encoding anti-PD-L1 scFv-Fc fused with targeting elements of MIC5 (te MIC5) comprising the signal sequence and propeptide of MIC5. The scFv portion composed of VH and VL and fused with CH2 and CH3 portion of murine IgG2a (mIgG2a). (B) After complete lysis of HFF cells infected with Nc-1-scFv-Fc or Nc-1 supernatants (S Nc-1-scFv-Fc and S Nc-1) and enriched supernatant (SE Nc-1-scFv-Fc and SE Nc-1) were analyzed by western blot. scFv-Fc (CHO) corresponds to purified scFv-Fc produced in CHO cells. (C) HFF cells infected with Nc-1 or Nc-1-scFv-Fc were stained with anti-MIC3 (red) and anti-mouse IgG (blue) and individual intracellular Nc-1-scFv-Fc are observed according to GFP expression (green). Overlapping of red and blue signals resulted in merge images (pink). (D) Supernatants were collected at different times and concentrations of scFv-Fc were reported in nanogram per 10 7 tachyzoites. (E and F) Temperature and ethanol influence on scFv-Fc secretion. Extracellular Nc-1-scFv-Fc were incubated at different temperatures (E) or in 1% ethanol (F). Condition at 37°C and without ethanol were respectively designated as reference (100%). For (D), (E), and (F), data are expressed as mean values ±SD ( n = 3); significant differences are indicated as ∗ p < 0.05).

Article Snippet: For subcellular localization of scFv-Fc in recombinant N. caninum , indirect immunofluorescence assays were performed on intracellular tachyzoites grown overnight in HFF cells on coverslip (Fisher Scientific) at MOI 1.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Lysis, Infection, Western Blot, Purification, Produced, Staining, Expressing, Incubation

Journal: Molecular Therapy Oncology

Article Title: Neospora caninum as delivery vehicle for anti-PD-L1 scFv-Fc: A novel approach for cancer immunotherapy

doi: 10.1016/j.omton.2025.200968

Figure Lengend Snippet: Infectious capacity of Nc-1-scFv-Fc evaluated in invasion assay (A) Attached extracellular tachyzoites (yellow) and intracellular tachyzoites (green) were labeled with anti-Nc-1. Host cell nuclei were stained with Hoechst (blue). (B) Attached extracellular and intracellular tachyzoites (total) per 100 HFF cells and (C) intracellular tachyzoites indicated as percentages based on total tachyzoites. Total numbers and intracellular tachyzoites were counted for five randomly selected fields of each strain. (B) and (C) are representative of three independent experiments with similar results. ns: not significant.

Article Snippet: For subcellular localization of scFv-Fc in recombinant N. caninum , indirect immunofluorescence assays were performed on intracellular tachyzoites grown overnight in HFF cells on coverslip (Fisher Scientific) at MOI 1.

Techniques: Invasion Assay, Labeling, Staining

Journal: Molecular Therapy Oncology

Article Title: Neospora caninum as delivery vehicle for anti-PD-L1 scFv-Fc: A novel approach for cancer immunotherapy

doi: 10.1016/j.omton.2025.200968

Figure Lengend Snippet: Induction of phagocytosis and ADCC with scFv-Fc secreted by Nc-1-scFv-Fc (A) RAW-264.7 cells were incubated with culture supernatant from HFF cells alone (Ctrl) or infected with Nc-1 (S Nc-1), Nc-1-scFv (S Nc-1-scFv-), or Nc-1-scFv-Fc (S Nc-1-scFv-Fc). Bound antibody fragment was detected using protein L (PpL_PE). (B) B16F10 cells expressing mCherry were co-cultured with Raw-264.7 at a ratio of 1:1. Raw 264.7 in culture supernatant of Nc-1 alone (S Nc-1), S Nc-1 complemented with purified scFv-Fc from CHO cells (Nc-1+scFv-Fc [CHO]), of Nc-1-scFv (S Nc-1-scFv) or of Nc-1-scFv-Fc (S Nc-1-scFv-Fc). The level of phagocytosis was defined with double-positive events (FITC+/mCherry+). (C) ADCC Reporter Bioassay performed with culture supernatants of either Nc-1 (S Nc-1), Nc-1-scFv (S Nc-1-scFv), or Nc-1-scFv-Fc (S Nc-1-scFv-Fc). Luminescence was measured in relative light units. Data are expressed as mean values ±SD ( n = 3; significant differences are indicated as ∗ p < 0.05; ∗∗ p < 0.01).

Article Snippet: For subcellular localization of scFv-Fc in recombinant N. caninum , indirect immunofluorescence assays were performed on intracellular tachyzoites grown overnight in HFF cells on coverslip (Fisher Scientific) at MOI 1.

Techniques: Incubation, Infection, Expressing, Cell Culture, Purification, Bioassay