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International Journal of Bioprinting Aflatoxin B1-induced cancer stem cells

the most significant in cancer pathogenesis . However, its tendency proved that single tumor spheroid imaging,
[63]
role as a CSC inducer has not been thoroughly investigated. like the frequently used FACS analysis, could also be an
Kawasaki et al. isolated hepatoma K2 cells from rat effective method for investigating the correlation between
exposed to aflatoxin B1 . They found that 89% of cell liver CSCs and the carcinogen aflatoxin B1. Furthermore, it
[64]
population was occupied by CSCs, while stemness genes should be noticed that single tumor spheroids for imaging
such as sox2, nanog, and klf4 were remarkably expressed. and FACS analysis were fabricated by 3D bioprinting. The
In addition, by treating HepG2 cells with various aflatoxin present study clearly demonstrated the possibility of drug-
B1 concentrations and sorting out CSCs based on surface resistant CSCs forming in a single liver tumor spheroid
markers, Ju et al. confirmed a similar result regarding by exposure to aflatoxin B1 carcinogen. A single tumor
the ability of aflatoxin B1 ability to induce CSCs . spheroid can correspond to a human patient having liver
[18]
Nonetheless, both aforementioned findings were observed tumor based on the fact that the patient may be exposed to
in 2D cell culture, which has been proven to have numerous aflatoxin B1 through daily diet.
limitations, making it clinically less significant. This study,
which examines the phenomena in 3D tumor spheroid, may 5. Conclusion
offer a novel and more remarkable insight into the actual
effects of aflatoxin B1 on the formation of CSCs in a tumor. In the present study, we developed a 3D tumor spheroid
model fabricated via 3D bioprinting to monitor the formation
In this study, two methods were employed to determine of liver CSCs induced by aflatoxin B1. CSCs were observed
the presence of CSCs. Regarding single spheroid imaging, using single tumor spheroid imaging and FACS analysis.
the analysis at the single-cell level indicated that the higher After using both methods, a correlation between aflatoxin B1
the aflatoxin B1 concentration for treatment, the lower the dose and CSC level was observed: the higher the aflatoxin B1
cell number in a spheroid. This could be attributed to the concentration for treatment, the higher the number of CSCs
dual influence exerted by aflatoxin B1. Specifically, aflatoxin could be detected. The similarity between the detailed results
B1 not only induces CSCs but also causes DNA damage. This of two methods suggests the suitability of 3D bioprinting-
phenomenon is more evident at higher concentrations . based single tumor spheroid analysis for quantification of
[65]
This could also explain the deviation between the results drug-resistant single liver CSCs induced by aflatoxin B1.
obtained from single spheroid imaging and FACS in the
present study. In the results of single spheroid imaging Acknowledgments
and FACS, similarities could be observed in the control We are grateful to the Research Institute of Pharmaceutical
and low dose-treated samples. However, the difference Sciences at Seoul National University for providing
in the number of CSCs acquired by FACS and imaging experimental equipment and Brain Korea 21 Plus (BK21
became more noticeable as the concentration of aflatoxin Plus).
B1 increased. Indeed, as the tumor spheroids had a smaller
number of single cancer cells, the percentage of single CSCs Funding
obtained by the spheroid imaging showed higher values of
19.2% and 29% at aflatoxin B1 concentrations of 2.5 and This work was supported by a National Research Foundation
5 μM, compared to 15% and 20.3%, respectively, acquired by of Korea (NRF) grant funded by the Ministry of Education,
FACS analysis at identical aflatoxin B1 concentrations. This Science and Technology (MEST) (No. 2018M3A7B4071235,
is attributed to the growth of single tumor spheroids being 2023R1A2C3006991, and RS-2023-00218543).
significantly inhibited by the DNA-damaging aflatoxin B1,
and this phenomenon occurs more frequently at higher Conflict of interest
concentrations of aflatoxin B1. As a result, Table 2 shows The authors declare no conflicts of interests.
that there was smaller population of single cancer cells in
single spheroids at higher concentrations of aflatoxin B1. In Author contributions
addition, owing to the growth-inhibiting effect of aflatoxin Conceptualization: Joon Myong Song
B1, more single cancer cells fixed in the hydrogel were Formal analysis: Viet Phuong Cao, Sera Hong
observed. They could not be distinguished and excluded Investigation: Viet Phuong Cao
through the FACS analysis, although they could contribute Supervision: Joon Myong Song
to the entire population, regardless of which markers Writing – original draft: Viet Phuong Cao
they expressed. However, it was obvious that the increase Writing – review & editing: Viet Phuong Cao, Joon Myong
in the number of drug-resistant CSCs as the aflatoxin B1 Song, Sera Hong
concentration increased showed an identical tendency in
both spheroid imaging and FACS analysis. This similar All authors approved the final version of the manuscript.

Volume 9 Issue 6 (2023) 369 https://doi.org/10.36922/ijb.0985