br The understanding of the heterogeneity

The understanding of the heterogeneity of tumor CFTRinh-172 composing the tumor tissue is of great importance for the treat-ment of malignant tumors [10]. Populations of tumor cells with different phenotypic characteristics exist within the tumor mass, and they are sometimes diffusely combined or intermingled [11,12].
Different subpopulations of tumor cells in the tumor tissue provide different sensitivities to therapeutic agents, leading to the selection of surviving cells that are resistant to drugs [13,14]. Moreover, epithelial-mesenchymal transition can also generate many variants of tumor cells [15,16]. If the cancer stem cell theory is applicable for the development of malignant tumors, it would indicate that the tumor cells with variable phenotypes can differentiate from the cancer stem cells through non-symmetrical cell division [17,18].
Although Kras mutations are a pivotal factor for the develop-ment of pancreatic cancer [19], the heterogeneity of pancreatic cancer cells with different phenotypes has also been reported [20]. If pancreatic cancer tissues contain a clonal subpopulation of cells that are highly proliferative, these cells would dominate the tumor, resulting in aggressive disease. The identification of genes responsible for conferring such malignant phenotypes is essential because the inhibitors of these gene products can become prom-ising therapeutic modalities for molecular targeted therapy.
CLDN family are important components of tight junctions regulating paracellular permeability, cell polarity and barrier function permanence [21,22], and are closely associated with ma-lignant phenotypes [23,24]. CLDN-associated cell-to-cell adhesion controlling luminal barrier, paracellular transport and signal transduction is an important factor for the proliferation, trans-formation and metastasis of tumor cells [23]. Although the expression of CLDN in human pancreatic cancer tissue has been widely investigated [25e28], the involvement of CLDN7 in human pancreatic cancer has not yet been defined [29e32].
In this study, we isolated several clonal populations represent-ing aggressive and non-aggressive phenotypes from a human pancreatic cancer cell line and subsequently identified CLDN7 as a gene specifically expressed in the clonal population with aggressive phenotypes.
Methods
Cell culture
The human pancreatic adenocarcinoma cell line, MIA PaCa-2, was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The MIA PaCa-2 cells were maintained in Dulbecco's modified Eagle's medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Biosera, Kansas City, MO, USA) and penicillin/streptomycin/Amphotericin B (Nacalai Tesque). The cells were cultured at 37 C in a fully humidified at-mosphere with 5% CO2. Other human pancreatic cancer cell lines, namely Panc-1, BxPC, AsPC, Capan-1 and Capan-2, and a human breast cancer cell line, MCF7 (positive control for CLDN7 expres-sion), were purchased from ATCC.
Isolation of several cell subpopulations derived from single MIA PaCa-2 cells
The limiting dilution method was used for the isolation of single cell clones from the MIA PaCa-2 cells. Single MIA PaCa-2 cells were placed in wells of culture plates, and the clonal populations growing from each single cell were isolated. Subpopulations of cells showing epithelial or non-epithelial like morphology were isolated, and single cell-derived clones were again generated from these subpopulations.
DNA short tandem repeat (STR) analysis
DNA STR analysis was performed at the JCRB Cell Bank of the National Institute of Biomedical Innovation (Osaka, Japan). The data from the DNA STR analysis of the MIA PaCa-2-A cells and MIA PaCa-  2-R cells were compared with those of the MIA PaCa-2 cells regis-tered in ATCC (CRL-1420 MIA PaCa-2) and JCRB (JCRB0070 MIA PaCa-2).