Page 16 - Read Online

Page 336 Seno et al. Cancer Drug Resist 2019;2:335-50 I http://dx.doi.org/10.20517/cdr.2019.01

Keywords: Daunorubicin, cancer stem cell, DNA fragmentation

INTRODUCTION
Cancer stem cells (CSCs) are considered to be responsible for various pathological phenomena in cancer,
[1,2]
such as tumor initiation, growth, tumor vascularization, recurrence, and metastasis . Multiple studies
have consistently reported that the higher resistance capacity of CSCs against chemo- and/or radio-therapies
compared to those of non-stem cancer cells significantly contributes to CSC survival after treatment. The
mechanisms underlying CSC-mediated resistance have been explained from various points of view, such as
the expression of multi drug antiporters and slower cell cycle progression of CSC (quiescent/dormant states)
[3]
in tumors . Recent studies have revealed that the tumor microenvironment also plays important roles in
the development of resistance to therapy . Thus, further understanding not only the nature of CSC itself
[4,5]
but also the relationship between CSCs and their microenvironment is required for the development of
[1,6]
treatment strategies that can effectively eliminate CSCs from tumors .
Given that CSC properties are regulated by the CSC niche, drug screening should be carried out in the
presence of CSC niches. However, the maintenance and amplification of CSCs and the reconstruction
[7]
of their niche in vitro are challenging when conducting large-scale drug screening . Recently, we have
established mouse induced pluripotent stem cells (miPSCs) that possess self-renewal and differentiation
capacities, as well as malignant tumorigenicity, which is in strong agreement with the definition of CSC [8-10] . These
miPSCs-derived CSCs (miPS-CSCs) could be maintained in vitro under conditions that allow spontaneous
differentiation. The miPS-LLCcm cell line (miPSC-CSC differentiated with Lewis lung carcinoma
conditioned medium) has been demonstrated to exhibit self-renewal ability by treatment with factors
secreted not only autocrinally from the CSCs, but also paracrinally from the differentiated progeny of miPS-
[11]
LLCcm cells . Notch signaling in self-renewing CSCs of miPS-LLCcm is likely to be activated by factor(s)
secreted from the endothelial-like cells that were differentiated from CSCs of miPS-LLCcm. Additionally, we
reported that culturing miPS-LLCcm cells without the differentiated population influenced the endothelial
differentiation capacity of CSCs, thereby suggesting that the signal(s) from the endothelial cells contribute to
endothelial differentiation of CSCs [11,12] . Thus, we concluded that the generated miPS-CSCs established their
niche in vitro by autonomously sustaining self-renewal and differentiation, consistent with the endothelial/
perivascular niche of CSCs [13-17] . miPS-CSCs could serve as an advanced model for the development of anti-
CSC drugs.

In the present study, we performed drug screening to identify effective drugs against CSCs using miPS-
CSCs, the miPS-derived models of CSCs. In addition we examined the effectiveness of the drug candidates,
especially the apoptotic pathway induced by the anthracycline daunorubicin in miPS-LLCcm.

METHODS
Cell culture
MiPSCs (iPS-MEF-Ng-20D-17) were obtained from the RIKEN Cell Bank and maintained on mitomycin
[18]
C-treated MEF on gelatin-coated dishes in miPS medium containing Dulbecco’s modified eagle’s medium-
high glucose (DMEM, Sigma, MO), 15% FBS (Gibco, NY), 0.1 mmol/L non-essential amino acids (Gibco,
NY), 2 mmol/L L-glutamine (Nacalai Tesque, Japan), 0.1 mmol/L 2-mercaptoethanol (Nacalai Tesque,
Japan), 50 U/mL penicillin (Nacalai Tesque, Japan), 50 µg/mL streptomycin (Nacalai Tesque, Japan), and
1000 U/mL Leukemia inhibitory factor (LIF, Millipore, MA). The miPS-CSCs were maintained in miPS
media without LIF on gelatin-coated dishes . Mouse Lewis lung carcinoma (LLC) cells, mouse normal
[8,9]
fibroblast BALB/c 3T3 A31-714 C4 cells, and human cervical cancer Hela cells were maintained in DMEM