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. 2021 Jan 4;10:e55117. doi: 10.7554/eLife.55117

Figure 6. Dclk1+pancreatic ductal adenocarcinoma (PDAC) cells have remarkable spheroid- and tumor-forming potential.

Figure 6.

(A) Scheme of FACS and 3D culture of Dclk1+ and Dclk1− PDAC cells from DKPF mice. (B) FACS-sorting of PDAC cells on the basis of Epcam and Dclk1 expression in DKPF mice. (C and D) Representative images of tumor spheroids derived from sorted Dclk1− (C) and Dclk1+ (D) PDAC cells at day 7. (E) Size of spheroids were compared (mean ± SEM; Dclk1+, n = 5; Dclk1−, n = 5; n: number of mice). Statistical significance of the differences is indicated as ***p<0.001, Student’s t-test. (F) The number of spheroids larger than 20 µm were compared (mean ± SEM; Dclk1+, n = 5; Dclk1−, n = 5; n: number of mice). Statistical significance of the differences is indicated as ***p<0.001, Student’s t-test. (G and H) Tumor-forming assay of Dclk1+ or Dclk1− PDAC cells. (G) Macroscopic image of the NOD/SCID mice after subcutaneous transplantation of Dclk1+ or Dclk1− PDAC cells into the flank. A thousand of Dclk1+ PDAC cells developed subcutaneous tumors whereas Dclk1− PDAC cells did not. (H) Dilution series showed 100, 500, or 1000 Dclk1+ PDAC cells developed subcutaneous tumors in 16.7%, 25.0%, and 75.0% of NOD/SCID mice, whereas same or larger numbers of Dclk1− PDAC cells did not. Statistical significance of the differences is indicated as p<0.05, chi-squared test. (I–K) Histological analysis of primary xenografts derived from FACS-sorted Dclk1+ PDAC cells. (I) Hematoxylin and Eosin (H and E) staining. (J) Immunostaining for Krt19. (K) Immunostaining for Dclk1. Scale bar, 200 µm (I), 50 µm (J and K). (L) Increasing curve of subcutaneous tumor (mean ± SEM; Dclk1+, n = 3; Dclk1−, n = 4; n: number of mice). Statistical significance of the differences is indicated as *p<0.05, Student’s t-test. (M) Passaged tumor-forming assay of Dclk1+ or Dclk1− xenograft cells collected by FACS. A thousand of Dclk1+ xenograft cells developed subcutaneous tumors whereas Dclk1− xenograft cells did not. (N–P) Histological analysis of passaged xenografts derived from FACS-sorted Dclk1+ primary xenograft cells. (N) Hematoxylin and Eosin (H and E) staining. (O) Immunostaining for Krt19. (P) Immunostaining for Dclk1. Scale bar, 200 µm (N), 50 µm (O and P).

Figure 6—source data 1. Growth of pancreatic ductal adenocarcinoma (PDAC) xenograft.
Measured value of increasing curve of subcutaneous tumor derived from Dclk1+ PDACs cells sorted by FACS.