Fig. 4.
In vitro MB cell line derived from Kif3a Math1-CreER, Kif3afl/fl, Ptch+/− mouse is tumorigenic. (A) MTT survival assay-derived cell growth ratio showing rapid proliferation of Math1-CreER, Kif3afl/fl MB cell line relative to both fibroblast and colon carcinoma cell lines. Error bars depict ±SE. (B) Math1-CreER, Kif3afl/fl MB cells form cilia under tumor ciliation protocol from Figure 3A. Percent ciliation is (number of cilia/number of nuclei) for each field and results depict quantification from three independent experiments. Percent ciliation of Ptch+/− MB is also shown for comparison. Error bars depict ±SE. (C) Immunostaining (×40) showing Math1-CreER, Kif3afl/fl MB cilia. Nuclei stained with DAPI (blue), cilia highlighted by acetylated tubulin (cyan). (D) Math1-CreER, Kif3afl/fl MB cells exhibit anchorage-independent growth in soft agar assay. Bar graph depicts number of colonies >2mm per ×10 field at 18 days after plating. Fibroblast cell line does not form colonies, but colon carcinoma and Math1-CreER, Kif3afl/fl MB cells readily form colonies in soft agar. Error bars depict ±SE. (E) Math1-CreER, Kif3afl/fl MB cells form heterotopic solid tumors when injected into the flank of nude mice. Tumor volume in cubic inches shown as a function of time (days) ofmeasurement after initial palpation of tumor. H&E image (×20) shows flank tumors with abundant cells with prominent nuclei in histological pattern consistent with MB. Error bars depict ±SE. (F) Math1-CreER, Kif3afl/fl MB cells injected into the cerebellum form solid tumors within 4–8 weeks. Left panel is a gross image of orthotopic tumor in central vermis region of cerebellum (encircled in red). Low power (×10, middle panel) shows infiltration of tumor cells within the cerebellum and high power (×30, far right panel) with line indicating the margin between normal cerebellum and MB. Tumor cells obliterate the left folium of the cerebellum (labeled MB) and a normal, organized cerebellum folium (labeled CB) is depicted on the right for comparison.