Fig 2: DGCs promote macrophage infiltration and tumor progression in cooperation with GSCs. a Flow cytometric analysis of CD133 in MGG8 cells. Sorted CD133 + cells were cultured as GSCs and CD133-cells were cultured as DGCs. b Representative images of paired GSCs and DGCs derived from two primary human GBM specimens (MGG4 and MGG8). Scale bar, 300 µm. c Representative image (left panel) and quantification (right panel) of transwell analysis of U937 macrophages upon stimulation with control medium or conditioned medium (CM) from DGCs (MGG4 and MGG8). Scale bar, 100 μm. n = 4 biological replicates, mean ± SEM, ***P < 0.001, Student’s t-test. d Kaplan–Meier (upper) and log-rank P value (bottom) analyses of mice bearing orthotopic xenografts of 1 × 103 glioma stem-like cells (GSCs) alone, 1 × 105 differentiated glioblastoma cells (DGCs) alone, or cotransplanted 1 × 104 DGCs with 1 × 103 GSCs derived from MGG8 cells. e Representative H&E stainings of tumor-bearing brains harvested at 30 days after implantation of 1 × 103 GSCs alone, or 1 × 103 GSCs plus 1 × 104 matched DGCs derived from MGG8. Scale bars indicate 2000, 100 and 50 µm in gross and detail views, respectively. Middle and right panels are high magnifications of the areas marked by rectangles in left panels. f Representative confocal images of tumor-bearing brains harvested at 30 days after implantation of 1 × 103 GSCs alone, or 1 × 103 GSCs plus 1 × 104 matched DGCs derived from MGG8. Scale bar, 100 µm. Iba1 (red), CD206 (green), and DAPI (blue). g Quantitation of pan-macrophage (Iba1+) and M2 macrophage (CD206+) densities in xenografts formed by 1 × 103 GSCs alone or 1 × 103 GSCs plus 1 × 104 matched DGCs derived from MGG8 cells. The total number of macrophages was counted in five randomly selected fields per sample. n = 5 biological replicates, mean ± SEM, ***P < 0.001, ****P < 0.0001, Student’s t-test. h Quantitation of the fraction of M2 macrophages (CD206+). The fraction was determined by M2 macrophages (CD206+) among pan-macrophages (Iba1+) in 1 × 103 GSCs alone or 1 × 103 GSCs plus 1 × 103 matched DGCs xenografts. The total number of macrophages was counted in five randomly selected fields per sample. n = 5 biological replicates. Data are represented as means ± SEM. **P < 0.01, Student’s t-test

Fig 3: CDKAL1 is highly expressed in rhabdomyosarcoma (RMS) cancer stem-like cells (CSCs). A) The human RMS cell line RD was fractionated by FACS according to CD133 expression level. The level in each fraction was confirmed by quantitative PCR (n = 2, error bars indicate mean ± SD). B,C) The self-renewal (B, n = 4) and colony-forming (C, n = 3) capacities of CD133high and CD133low RD cells (error bars indicate mean ± SD). D) Quantitative PCR for the expression profiling of tRNA-modifying enzymes was performed in the CD133high and CD133low RD cells. (E) Western blotting analysis to validate the elevated expression level of CDKAL1 in the CD133high RD cells. Note that this population harbored higher CSC-related potential than the CD133low RD cell population (Figure 1B,C). F) Immunofluorescent analysis using anti-CDKAL1 and anti-CD133 antibodies in RD (left) and RH30 (right) cells. Scale bars, 50 µm. G) Immunofluorescence analysis of normal skeletal muscle and pathological specimens of alveolar RMS (ARMS) and embryonal RMS (ERMS) using anti-CDKAL1 and anti-CD133 antibodies. Scale bars, 20 µm. H) Comparison of CDKAL1 expression in patients with fusion-negative and fusion-positive RMS in the GSE108022 dataset. I) The positive correlation between the expression levels of CD133 and those of CDKAL1 in a gene set of RMS patients (GSE108022). J) Kaplan-Meier curve displaying the prognosis in RMS patients with CDKAL1high and CDKAL1low signature from the E-TABM-1202 dataset. K) Gene set enrichment analysis (GSEA) displaying the stem cell-related gene sets that are enriched in the CDKAL1high RMS population in the E-TABM-1202 dataset.

Fig 4: CDKAL1 promotes the maintenance of CSC-related traits in RMS. A) The self-renewal capacity of Control-shRNA- or CDKAL1-shRNA-expressing RMS cell lines was measured (n = 4, error bars indicate mean ± SD). Western blotting results are shown to confirm the CDKAL1 knockdown. B) The clonogenic growth potential of Control-shRNA- or CDKAL1-shRNA-expressing RMS cells (n = 3, error bars indicate mean ± SD). C) The cell proliferation analysis of Control-shRNA- or CDKAL1-shRNA-expressing RD and RH30 cells (n = 4, error bars indicate mean ± SD). D) Flow cytometry analysis showing the effect of CDKAL1 knockdown on CD133 expression in RD cells and RH30 cells. E) Immunofluorescent imaging of myogenic differentiation of RMS cells after CDKAL1 knockdown, as indicated by positive staining for myosin heavy chain (MyHC). F) One million Control-shRNA- or CDKAL1-shRNA-expressing RMS-YM cells were subcutaneously injected at the backs of BALB/c-nu/nu mice, and tumor growth was measured. Ten injection sites were measured for each group (error bars indicate mean ± SD). G) Immunofluorescent analysis using anti-CD133 and anti-MyHC antibodies in the RMS-YM xenografts obtained in Figure 2E. All scale bars, 50 µm. H) Effect of the forced expression of CDKAL1 on the sphere-forming capacity of RD and ICH-ERMS-1 cells (n = 4, error bars indicate mean ± SD).

Fig 5: SALL2 is an essential factor for maintaining CSCs in RMS and is controlled by CDKAL1 at the translation initiation phase. A,B) The self-renewal capacity (A, n = 4, error bars indicate mean ± SD) and colony-forming potential (B, n = 3, error bars indicate mean ± SD) of Control-shRNA- or SALL2-shRNA-expressing RMS cells were measured (n = 4, error bars indicate mean ± SD). Western blotting results are shown to confirm SALL2 knockdown (A). C) One million Control-shRNA- or SALL2-shRNA-expressing RMS-YM cells were injected subcutaneously at the backs of BALB/c-nu/nu mice, and tumor growth was measured. Ten injection sites were measured for each group (error bars indicate mean ± SD). D) Immunofluorescent analysis using anti-CD133 and anti-MyHC antibodies in the RMS-YM xenografts obtained in Figure 5C. All scale bars, 50 µm. E) Immunofluorescent analysis of normal and pathological specimens using anti-CDKAL1 and anti-SALL2 antibodies. Note that CDKAL1high tumor cells exhibit SALL2high properties in RMS. Scale bars, 100 µm. F) Luciferase reporter analysis with 5’UTR-SALL2, -GAPDH, and -ACTB in Control-shRNA- or SALL2-shRNA-expressing RD cells (n = 3, error bars indicate mean ± SD). G) A cartoon representing the procedure. H) In situ proximity ligation assay detecting a signal obtained by the reactions from endogenous CDKAL1 and the DIG-labeled sense or antisense RNA probes of the 5’UTR region of SALL2 mRNA in Control-shRNA- or SALL2-shRNA-expressing RD cells. Scale bars, 10 µm. I) RNA immunoprecipitation and reverse transcription PCR assay using RD cells expressing myc-tagged CDKAL1. Primer pairs for 5’UTR of SALL2 mRNA were used to detect the interaction between CDKAL1 and SALL2 mRNA. Reverse transcriptase (-) samples serve as negative controls. Three samples (#1, #2, and #3) were analyzed.