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. 2023 Apr 15;24(8):7331.
doi: 10.3390/ijms24087331.

Identification and Clinical Significance of Pancreatic Cancer Stem Cells and Their Chemotherapeutic Drug Resistance

Yu-Chi Kuo  1 , Hao-Wei Kou  1 , Chih-Po Hsu  1 , Chih-Hong Lo  1 , Tsann-Long Hwang  1
Affiliations

Identification and Clinical Significance of Pancreatic Cancer Stem Cells and Their Chemotherapeutic Drug Resistance

Yu-Chi Kuo et al. Int J Mol Sci. .

Abstract

Pancreatic cancer ranks in the 10th-11th position among cancers affecting men in Taiwan, besides being a rather difficult-to-treat disease. The overall 5-year survival rate of pancreatic cancer is only 5-10%, while that of resectable pancreatic cancer is still approximately 15-20%. Cancer stem cells possess intrinsic detoxifying mechanisms that allow them to survive against conventional therapy by developing multidrug resistance. This study was conducted to investigate how to overcome chemoresistance and its mechanisms in pancreatic cancer stem cells (CSCs) using gemcitabine-resistant pancreatic cancer cell lines. Pancreatic CSCs were identified from human pancreatic cancer lines. To determine whether CSCs possess a chemoresistant phenotype, the sensitivity of unselected tumor cells, sorted CSCs, and tumor spheroid cells to fluorouracil (5-FU), gemcitabine (GEM), and cisplatin was analyzed under stem cell conditions or differentiating conditions. Although the mechanisms underlying multidrug resistance in CSCs are poorly understood, ABC transporters such as ABCG2, ABCB1, and ABCC1 are believed to be responsible. Therefore, we measured the mRNA expression levels of ABCG2, ABCB1, and ABCC1 by real-time RT-PCR. Our results showed that no significant differences were found in the effects of different concentrations of gemcitabine on CSCs CD44+/EpCAM+ of various PDAC cell line cultures (BxPC-3, Capan-1, and PANC-1). There was also no difference between CSCs and non-CSCs. Gemcitabine-resistant cells exhibited distinct morphological changes, including a spindle-shaped morphology, the appearance of pseudopodia, and reduced adhesion characteristics of transformed fibroblasts. These cells were found to be more invasive and migratory, and showed increased vimentin expression and decreased E-cadherin expression. Immunofluorescence and immunoblotting experiments demonstrated increased nuclear localization of total β-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells showed activation of the receptor protein tyrosine kinase c-Met and increased expression of the stem cell marker cluster of differentiation (CD) 24, CD44, and epithelial specific antigen (ESA). We concluded that the expression of the ABCG2 transporter protein was significantly higher in CD44+ and EpCAM+ CSCs of PDAC cell lines. Cancer stem-like cells exhibited chemoresistance. Gemcitabine-resistant pancreatic tumor cells were associated with EMT, a more aggressive and invasive phenotype of numerous solid tumors. Increased phosphorylation of c-Met may also be related to chemoresistance, and EMT and could be used as an attractive adjunctive chemotherapeutic target in pancreatic cancer.

Keywords: ABCG2 transporter; CD44; EpCAM; cancer stem cells; chemoresistance; pancreatic ductal adenocarcinoma.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The survival of cancer stem cells CD44+/CD44− of BxPC-3 PDAC cells treated with or without gemcitabine (25 nM, 72 h) is shown. The CD44+ had better survival than CD44−, * < 0.05. (Mean ± SD).
Figure 2
Figure 2
The survival of cancer stem cells EpCAM+/EpCAM− of BxPC-3 PDAC cells treated with or without gemcitabine (25 nM, 72 h) is shown. The EpCAM+ had better survival than EpCAM−, * < 0.05. (Mean ± SD).
Figure 3
Figure 3
The survival of parental BxPC-3 PDAC cells treated with different concentrations of verapamil and gemcitabine is shown. Verapamil 25 and 50 µM exerted the best chemotherapeutic effects when gemcitabine concentration of 0.001 µg/mL was used, * < 0.05. (Mean ± SD).
Figure 4
Figure 4
The survival of CD44+ cancer stem cells of BxPC-3 PDAC cells treated with different concentrations of verapamil and gemcitabine is shown. Verapamil 25 and 50 µM exerted the best chemotherapeutic effects when gemcitabine concentration of 0.001 µg/mL was used, * < 0.05. (Mean ± SD).
Figure 5
Figure 5
The expression level of 4 chemoresistance genes for non-drug treatment CD44− cancer stem cells and CD44−BxPC-3 PDAC cells is shown. The ABCG2 gene had significantly higher expression in CD44+ than CD44−, * < 0.05. (Mean ± SD).
Figure 6
Figure 6
The expression level of 4 chemoresistance genes for non-drug treatment EpCAM+ cancer stem cells and EpCAM− BxPC-3 PDAC cells is shown. The ABCG2 gene had significantly higher expression in EpCAM+ than EpCAM−, * < 0.05. (Mean ± SD).
Figure 7
Figure 7
The response of 4 chemoresistance genes for CD44+ cancer stem cells and CD44−BxPC-3 PDAC cells treated with gemcitabine (25 nM, 48 h) is shown. The ABCG2 gene had significantly higher expression in CD44+ than CD44−, * < 0.05. (Mean ± SD).
Figure 8
Figure 8
The response of 4 chemoresistance genes for EpCAM+ cancer stem cells and EpCAM− BxPC-3 PDAC cells treated with gemcitabine 25 nM, 48 h is shown. The ABCG2 gene had significantly higher expression in EpCAM+ than EpCAM−, * < 0.05. (Mean ± SD).
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