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  • br To investigate whether the cell population of tumor

    2020-08-18


    To investigate whether the cell population of tumor-like co-spher-oids formed on CS-HA may be influenced by the ratio of MIA JSH 23 and PSCs seeded initially, the respective cell numbers and sizes of the formed spheroids were determined in the conditions of different cell ratios. As shown in Fig. S5A, the cell count of mono-cultured MIA cells on CS-HA was significantly increased 2.9-fold after 72 h. The cell count of mono-cultured PSCs on CS-HA was reduced after 24 h but increased
    Fig. 1. Morphological phenotype of pancreatic cancer cells grown on plates coated with different polymers [chitosan, chitosan-HA (CS-HA), and polyvinyl alcohol (PVA)] after 24 h and 96 h. Co-cultured pancreatic cancer cells (AsPC-1 or MIA) and the pancreatic stellate cells (PSCs) showed various extents of clustering and attachment on the chitosan plates, and formed tumor-like spheroids on chitosan-HA plates. The cells on PVA showed large aggregates. The AsPC-1 and MIA cells were labeled with PKH26 (red) while PSCs were labeled with PKH67 (green) fluorescence dyes as indicated. The scale bar represents 300 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    2.1-fold after 72 h. The total number of co-cultured MIA cells and PSCs at 24 h was significantly decreased as the cell ratio of PSCs in MIA cells increased (Fig. S5, SI). The population of MIA cells in the co-spheroids was elevated after 24 h, 48 h, and 72 h of co-culture. At a ratio where the PSCs dominated, the MIA cells grew as the major population of cells. After 72 h, the size of tumor-like spheroids was significantly in-creased ∼4.8-fold in the mono-cultured MIA group and ∼4-fold in co-cultured MIA:PSC (1:9) (Fig. S5B, SI). After 48 h, the diameter of tumor-like spheroids was around 190 μm in mono-cultured MIA and co-cul-tured MIA:PSC (1:9) groups. On the other hand, there was no significant change in the size and diameter of PSC homo-spheroids after 48 h (Fig. S5B and S5B, SI).
    3.2. Migratory behavior of tumor spheroids on CS-HA coated plates
    Real-time recording was used to monitor the formation of tumor 
    spheroids, and the spheroid-forming process is demonstrated in sup-plementary videos. The static time-lapse images of co-cultured MIA cells and PSCs on CS-HA coated plates between 0 h and 8 h after seeding are displayed in Fig. 3A. The tumor-like co-spheroidal structure gra-dually formed in 8 h. The mono-cultured MIA cells showed slow but persistent migration in the absence of PSCs (Video S1, SI), and they became aggregated, forming clusters in 48 h. The mono-cultured PSCs displayed higher migration activity and moved in a broad range and distance (Videos S2 and S3, SI). Meanwhile, the co-cultured MIA cells and PSCs were assembled into 3D co-spheroids with concentric core-shell structure. The cell distribution of MIA cells and PSCs of the spheroids is dependent on the co-culture ratios. The MIA cells were wrapped in the PSCs and fused into the spheroid in the 1:9 group (Video S7, SI). However, the MIA cells were randomly distributed in the outer layer of the spheroids in the 1:1 group (Video S5, SI). Judging from the real-time imaging, PSCs migrated to the neighborhood of MIA cells to
    Fig. 2. The morphology of pancreatic cancer associated cells cultured on the CS-HA plates for 24 or 48 h. The pancreatic cancer cells (MIA cells) were labeled with PKH26 (red) and the pancreatic stellate cells (PSCs) were labeled with PKH67 (green) fluorescence dyes as indicated. (A) MIA cells showed clustering on the CS-HA coated plates. (B) PSCs formed 3D spheroids on the CS-HA coated plates. (C) MIA cells and PSCs seeded in a ratio 1:9 also formed 3D spheroids on the CS-HA coated plates. Images of (A–C) were taken by the inverted fluorescence microscope. (D) Confocal images of tumor-like co-spheroid structure from MIA and PSCs seeded in different ratios (1:3, 1:5, and 1:9) on the CS-HA coated plates for 48 h. The cross-sectional images of the co-spheroid at the middle depth demonstrated that PSCs were located in the outer layer (red circles) and MIA cells were located in the core region. Spheroid fusion could occur (as shown). Z-stack images were collected at 5 μm sections. BF: bright field. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    (caption on next page)
    Fig. 3. Developmental dynamics of the tumor-like spheroids from MIA cells and PSCs. (A) Time-lapse images of co-cultured MIA cells (red color) and PSCs (green color) on the CS-HA coated plates. Images were taken from the time period between 0 h and 8 h after seeding. The tumor-like co-spheroid structure gradually formed in 8 h. Blue arrows represent the spheroid formation. The scale bar represents 100 μm. The trajectories of MIA cells and PSCs (B), 1:9 co-cultured cells (C) on the CS-HA coated plates. Images were obtained at 8 h after analyses by the image recognition software. (D) Averaged values for the mean velocities of MIA cells, PSCs, and co-cultured cells in different ratios (1:1, 1:3, 1:5, and 1:9) on the CS-HA coated plates in 8 h. Each dot represents the mean velocity of a single cell. The black horizontal line shows 95% confidence intervals of mean velocities and standard deviation. # indicate statistically significant differences, #, p < 0.05, ##,