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International Journal of Bioprinting Aflatoxin B1-induced cancer stem cells

Figure 2. Confocal images of single tumor spheroids treated with aflatoxin B1 concentration of (a) 0, (b) 1, (c) 2.5, and (d) 5 μM. The expression of CD133
and ALDH1 was indicated by green and red colors, respectively, while the nucleus of single cancer cells was indicated by blue color. (e) Fluorescence
intensities of CD133 and ALDH1 measured from the obtained images as a function of aflatoxin B1 concentration. All scale bars: 10 μm. **P < 0.01, ***P
< 0.001, ****P < 0.0001.

spheroids showed different growth rate; spheroid number HepG2 cell surface, while ALDH1 exists in the cytosol of
18 grew up the most significantly by more than 220%, HepG2 cells. Therefore, in HepG2 cells where both markers
whereas the least growing spheroid, numbered 12, grew up are found, an absolute overlap of these two markers cannot
by only 42% (Figure 1e). However, the difference in the size be detected. Instead, the fluorescence image of CD133
of spheroids was similar between the first day of 12.4 μm surrounds that of ALDH1. As revealed by the images of
(from 13.1 to 25.5 μm) and 7th day of 16.6 μm (from 27.2 single spheroids, in the control, the fluorescence images
to 53.7 μm). This result suggests that the developed 3D of CD133 and ALDH1 were shown in a small region of
bioprinting model is an appropriate platform for 3D tumor the single spheroids, whereas the fluorescent images were
spheroid culture, which enabled the cells to grow naturally, expanded to a much larger region of the single spheroids
thereby preserving the heterogeneity of the cell population. at an aflatoxin B1 concentration of 5 μM. In detail, the
Moreover, as revealed in Figure S1 in Supplementary File, average fluorescence intensities of two expressed markers
the cell viabilities of tumor spheroids maintained more were measured over the whole single spheroid surface.
than 90% throughout the measurement period. These Table 1 displays the fluorescence intensity of the obtained
results demonstrated that the developed model could keep images; both increased gradually from the control to the
the cells alive without any special intervention. aflatoxin B1 concentration of 5 μM. In comparison to the
control, wherein the average intensities of CD133 and
3.2. Single tumor spheroids analysis based on the ALDH1 were 11.6 and 14.5, respectively, the aflatoxin B1
expression of cancer stem cell markers concentration of 5 μM caused nearly three times increase
Figure 2 shows the immunostaining results of HepG2 in the average intensities of CD133 and ALDH1 (35.4
spheroids exposed to four different aflatoxin B1 and 43.7, respectively) (Figure 2e). The increment in
concentrations, i.e., 0, 1, 2.5, and 5 μM, on the 7th day. The fluorescence intensities of CD133 and ALDH1 markers
blue, green, and red signals indicate cell nuclei, CD133, revealed that the drug-resistant properties of single HepG2
and ALDH1 markers, respectively. The positivity of both tumor spheroids were enhanced as a result of aflatoxin B1
CD133 and ALDH1 markers was partially shown in single treatment.
cells of single HepG2 spheroids (Figure 2). The extents to
which they were expressed increased as the concentration The number of single CSCs formed by exposure
of treated aflatoxin B1 increased. CD133 is expressed on the to aflatoxin B1 was determined at the single tumor

Volume 9 Issue 6 (2023) 365 https://doi.org/10.36922/ijb.0985