Fig 2: 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 × 105 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.

Fig 3: 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.

Fig 4: 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.