recombinant human cxcl3 protein  (R&D Systems)

Journal: Brain Pathology

Article Title: Intracerebellar administration of the chemokine Cxcl3 reduces the volume of medulloblastoma lesions at an advanced stage by promoting the migration and differentiation of preneoplastic precursor cells

doi: 10.1111/bpa.13283

Figure Lengend Snippet: Intracerebellar treatment with Cxcl3 dramatically reduces lesion volume in 4‐month‐old Ptch1 + / − / Tis21 − / − mice. (A) Treatment timeline: 3‐month‐old Ptch1 + / − / Tis21 − / − mice were treated for 28 days with Alzet minipumps filled with recombinant Cxcl3 or vehicle (CSF); a single injection of BrdU was performed 5 days before immunohistochemical analysis. (B) MB lesion frequency and number of preneoplastic lesions per cerebellum in CSF‐treated and Cxcl3‐treated mice. The number of mice analyzed for each treatment and the statistical analysis are indicated. (C) Representative confocal images of cerebellar sagittal slices of 4‐month‐old Ptch1 + / − / Tis21 − / − mice treated with CSF (left) or Cxcl3 (right), respectively. Nuclei were stained with Hoechst 33258 and lesions were identified by the presence of BrdU + pGCPs (red). Scale bar, 500 μm. (D, E) Bar graphs show the mean ± SEM of lesion areas (D) and lesion volumes (E) measured at the end of 28 days of treatment with Cxcl3 or vehicle. * p < 0.05, **** p < 0.0001, Student's t test; mice analyzed: n = 8 for vehicle, n = 9 for Cxcl3.

Article Snippet: Scraped off cells were removed by two washes with PBS; fresh medium supplemented with 100 ng/ml of human Cxcl3 (277‐GG‐010/CF, R&D Systems) or vehicle alone (PBS, 0.1% BSA) was then added to each well, and the cells were maintained at 37°C and 5% CO 2 for the whole duration of the experiment.

Techniques: Recombinant, Injection, Immunohistochemical staining, Staining

Journal: Brain Pathology

Article Title: Intracerebellar administration of the chemokine Cxcl3 reduces the volume of medulloblastoma lesions at an advanced stage by promoting the migration and differentiation of preneoplastic precursor cells

doi: 10.1111/bpa.13283

Figure Lengend Snippet: In 4‐month‐old Ptch1 + / − / Tis21 − / − mice, treatment with Cxcl3 induces neoplastic precursors to migrate from MB lesions to the IGL and differentiate. (A) Experimental timeline: 3‐month‐old Ptch1 + / − / Tis21 − / − mice were treated for 4 weeks with Alzet minipumps filled with recombinant Cxcl3 or CSF alone; mice received a single injection of BrdU at P113 and were analyzed by immunohistochemistry at P118. (B) Representative confocal images of pGCPs migrating out of MB lesions, identified as BrdU + cells (red), in Ptch1 + / − / Tis21 − / − cerebella chronically treated with CSF or Cxcl3. Sections are counterstained with Hoechst 33258 to visualize the lesion (L), molecular layer (ML), and internal granular layer (IGL). Scale bar, 50 μm. (C) Migrating pGCPs were quantified as percentage ratio (mean ± SEM) of BrdU + cells present within the ML or IGL area adjacent to each lesion to the total number of BrdU + cells in the lesion, ML and IGL. ** p < 0.01, **** p < 0.0001, Student's t test; mice analyzed: n = 6 for each treatment. (D) The same confocal sections of (B) were also reacted with antibody against NeuN (green), to label migrating and differentiating pGCPs (BrdU + NeuN + ). Sections are counterstained with Hoechst 33258 to visualize the lesion, ML and IGL. Scale bar, 50 μm. Some differentiated cells in the IGL are indicated by white arrows. (E) Quantification of the percentage ratio (mean ± SEM) of BrdU + NeuN + cells present within the ML or IGL area adjacent to each lesion to the total number of BrdU + cells in the lesion, ML and IGL. * p < 0.05, **** p < 0.0001, Student's t test; mice analyzed: n = 6 for each treatment.

Article Snippet: Scraped off cells were removed by two washes with PBS; fresh medium supplemented with 100 ng/ml of human Cxcl3 (277‐GG‐010/CF, R&D Systems) or vehicle alone (PBS, 0.1% BSA) was then added to each well, and the cells were maintained at 37°C and 5% CO 2 for the whole duration of the experiment.

Techniques: Recombinant, Injection, Immunohistochemistry

Journal: Brain Pathology

Article Title: Intracerebellar administration of the chemokine Cxcl3 reduces the volume of medulloblastoma lesions at an advanced stage by promoting the migration and differentiation of preneoplastic precursor cells

doi: 10.1111/bpa.13283

Figure Lengend Snippet: Expression of the chemokine receptor Cxcr2 in MB tissue of Ptch1 + / − / Tis21 − / − mice and DAOY cells. (A) Representative confocal images of Cxcr2 + cells (red) in MB lesion (left) and tumor tissue (middle) of Ptch1 + / − / Tis21 − / − mice, and SHH‐type MB cell line DAOY (right). Sections are counterstained with Hoechst 33258 to visualize nuclei. Scale bar, 50 μm. Cells in boxed areas are shown at higher digital magnification (3×). (B) Test of the ability to migrate in response to Cxcl3 of DAOY cells by scratch wound healing assay. Representative photomicrographs of DAOY cells (magnification 4×; scale bar 500 μm) taken at time of scraping (0 h) and 24 h after treatment with 0.1% BSA (as control; top) or Cxcl3 (bottom). Dashed white lines indicate wound boundaries. (C) Mean ± SEM of migration rate calculated as reported in the Materials and Methods section 24 h after wound scratching in control or Cxcl3‐treated DAOY cells. (D) Quantification of migration rate, expressed as mean ± SEM, 24 and 48 h after wound scratching in DAOY cells treated with vehicle alone (white bars), with the chemokine Cxcl3 (black bars), with Reparixin (Rpx, red bars), or with the combination Cxcl3 + Rpx (blue bars), respectively. (C, D) Data were obtained from two separate experiments, counting approximately four fields per well (at least three wells per experiment). * p < 0.05, *** p < 0.001, Student's t test.

Article Snippet: Scraped off cells were removed by two washes with PBS; fresh medium supplemented with 100 ng/ml of human Cxcl3 (277‐GG‐010/CF, R&D Systems) or vehicle alone (PBS, 0.1% BSA) was then added to each well, and the cells were maintained at 37°C and 5% CO 2 for the whole duration of the experiment.

Techniques: Expressing, Wound Healing Assay, Control, Migration

Journal: Brain Pathology

Article Title: Intracerebellar administration of the chemokine Cxcl3 reduces the volume of medulloblastoma lesions at an advanced stage by promoting the migration and differentiation of preneoplastic precursor cells

doi: 10.1111/bpa.13283

Figure Lengend Snippet: In vivo imaging of an orthotopic xenograft mouse model obtained by intracerebellar implantation of engineered human DAOY MB cells. (A) Treatment timeline: Six/seven‐week‐old athymic nude mice ( Foxn1 nu / Foxn1 + ) were implanted into the left cerebellar hemisphere with 1 × 10 5 human DAOY MB cells engineered to express GFP and firefly Luciferase (FLuc) genes (D0); 14 days after cell grafting (D14), mice were grouped according to their bioluminescence values and implanted with Alzet minipumps filled with recombinant Cxcl3 or CSF. During 4 weeks of treatment, mice were imaged every 7 days (D21, 28, 35, and 42) via in vivo bioluminescent imaging to monitor tumor growth and spinal cord metastases. (B) Left: DAOY cells, infected with a lentiviral vector encoding GFP‐FLuc, express GFP in vitro as determined by confocal fluorescence imaging (green cells; nuclei were stained with Hoechst 33258). Middle: DAOY cells implanted in the cerebellum of nude mice give rise to large intracerebellar tumors, as visible by illuminating the explanted brains at the end of treatment with a “GFP flashlight” (Nightsea) which causes GFP + tumors to glow. Right: Representative image by confocal microscopy of a cerebellar sagittal slice obtained from the brain shown in the middle image, showing MB cells, identified as GFP + cells (in green), 42 days after intracerebellar injection of engineered DAOY cells. The section is counterstained with Hoechst 33258 to visualize the ML and granule neurons in the IGL. Scale bars, 100 μm (left), 1 cm (middle), and 600 μm (right). (C) Representative bioluminescence images of nude mice orthotopically injected with DAOY‐Luc MB cells and after 14 days implanted with Alzet minipumps filled with CSF or Cxcl3, respectively (7 mice for each treatment group). Images are shown for days 21, 28, 35, and 42 after injection of DAOY‐Luc cells. Three animals, one CSF‐treated mouse (#5) and two Cxcl3‐treated mice (#11 and #14), developed metastases during the 28 days of Alzet implantation. (D) Tumor growth according to quantified electron emission (e − /s) from the cerebellar region of mice injected with DAOY‐Luc cells and treated in vivo for 4 weeks with Cxcl3 (orange) or CSF as vehicle (blue). The filled circles and the error bars indicate the mean ± SEM of the bioluminescence imaging (BLI) increase measured at the days of treatment indicated with respect to the day of Alzet implantation (D14). p = 0.4124 at D21, p = 0.8462 at D28, p = 0.9867 at D35, p = 0.9500 at D42, Student's t test.

Article Snippet: Scraped off cells were removed by two washes with PBS; fresh medium supplemented with 100 ng/ml of human Cxcl3 (277‐GG‐010/CF, R&D Systems) or vehicle alone (PBS, 0.1% BSA) was then added to each well, and the cells were maintained at 37°C and 5% CO 2 for the whole duration of the experiment.

Techniques: In Vivo Imaging, Luciferase, Recombinant, In Vivo, Imaging, Infection, Plasmid Preparation, In Vitro, Fluorescence, Staining, Confocal Microscopy, Injection