Human GBC is a highly aggressive malignant tumor with special biological behavior and poor prognosis. Surgical resection, chemotherapy and radiotherapy for the disease are disappointing [
1‐
6]. So, the development of novel adjuvant therapies, potential anticancer agents or molecularly targeted therapeutics for human GBC on the base of comprehensive investigating the biological behaviors and metastatic mechanism are very necessary; and novel GBC cell lines which were used as ideal experimental models in vitro and in vivo are urgently developed. In present study, we firstly established a novel highly aggressive GBC cell line derived from primary tumor, TJ-GBC2.
Human GBC cell lines are relatively scarce. Nowadays, more than a dozen of GBC cell lines were available, including G-415, GBK-1, KMG-A, FU-GBC-1, FU-GBC-2, NOZ, PTHrP-GBK, GB-d1, TGBC1TKB, TGBC2TKB, OCUG-1, TYGBK-1, HAG-1, GBC-SD, SGC-996, EH-GB1 and EH-GB2 [
8‐
24]. Of these, most were derived from the metastatic lesions of GBC patients, such as NOZ, OCUG-1, FU-GBC-2 and EH-GB1 from the ascites or the abdominal wall [
11,
13,
21], TYGBK-1 from a lymph node [
24], and EH-GB2 from liver metastatic site [
23]; some were derived from primitive cultured tumor that planted in nude mice [
18]; whereas others had themselves features, for example, GBK-1 was derived from human colony stimulating factor-producing GBC [
9], KMG-A from AFP-producing GBC [
10], and PTHrP-GBK from parathyroid hormone-related peptide producing GBC [
16]. Indeed, it is much more difficult to generate a primary cultured GBC cell line from a primary tumor than from metastases and ascites. This is because there are more fibrous tissues in GBC lesions, and biliary obstruction and infection contaminated GBC specimens. It is well known that the cell lines derived from ascites or other metastatic sites, or primitive cultured xenograft in nude mice were at least limited in two respects: one limitation was these cell lines derived from metastatic site losing the properties possessed in primary tumor, and cell line monoclonality that could not reflect heterogenic properties of the pleomorphic type of GBC; another limitation was these cell lines derived from xenografts of nude mice with a part of the immune function still having stronger immune-resistance. Therefore, culture of primary tumor of GBC may be a better way to build a cell line so as to accurately reflect the characteristics of the primary tumor cells [
26]. In present study, we successfully established a novel GBC cell line (TJ-GBC2) from a Chinese patient with primary GBC, with retaining characteristic epithelial tumor morphology and phenotypic in consistent with primary GBC. Although the cell line appears to have no prominent capacities of proliferation and growth in vitro and in vivo, chromosome analysis presented abnormity in structure and number of chromosome, and most cells (about 80%) were hypertetraploid, which implied high malignant potential. Coexistence of polygon, fusiform, irregular shape cells further implied that the cell line was derived from multicenter or polyclone. Therefore, primary cultured TJ-GBC2 cell line derived from primary tumor of GBC may reflect more accurately the characteristics of the primary GBC cells. TJ-GBC2 cell line proved to be an efficient tool for further investigation of the metastatic mechanism and potential targeted therapy of human GBC.
Epithelial tumor characteristics include epithelial morphological features, positive expression of epithelial markers with negative mesenchymal expression, and positive expression of epithelial tumor markers. As showed in Figs.
1 and
3, TJ-GBC2 cell line has characteristic epithelial tumor phenotypes as well as above characteristic epithelial tumor morphology in consistent with primary GBC. As we know, CK7, CK8, CK19 and E-cadherin are special epithelial markers; vimentin is characteristic mesenchymal marker; whereas CA19-9 and AFP are epithelial tumor markers. Electrochemistry luminescence immunity analysis showed that levels of CA19-9 and AFP were significantly increased in the culture supernatant of TJ-GBC2 cells; that CK7, CK8, CK19 and E-cadherin proteins were positively expressed in the xenograft of nude mice, with negative expression of mesenchymal marker vimentin, which is in accord with the results of primary tumor. These results verified TJ-GBC2 is an epithelial original cell line.
Metastasis, the spread of malignant cells from a primary tumor to distant sites and forms a tumor of same nature [
27,
28], is the biggest problem to cancer treatment [
29]. Abilities of migration and aggression affect the invasion and metastasis of tumor cells to a large extent. Intractability of gallbladder cancer also attribute to its early invasion and metastasis. In present study, we detected the aggressive and migration capabilities of five GBC cell lines, and expression of metastatic-related marker nm23 and MMP9 in the xenograft of TJ-GBC2 cell lines in nude mice. The results showed that TJ-GBC2 cell line had higher invasion ability and the highest migration ability compared to other human GBC cell lines such as GBC-SD and NOZ; and that MMP9 and nm23 were positively expressed in xenograft of nude mice, which is consistent with result of primary tumor of human GBCs. It was reported that GBC-SD is so far a human GBC cell line having the highest aggressive capability, which was derived from primary GBC [
15,
20,
22]; whereas NOZ is a human GBC cell line having the highest aggressive capability, which was derived from metastatic site of primary GBC [
11,
30]. In this study, cells that invaded through the basement membrane for 24 h having more than double cell number/fold in invasive assay and cells that were in a wound healing experiment for 24 h having completely healed cell wound were was used to defined as highly aggressive cell line. Considering GBC-SD and NOZ as the highest aggressive capability GBC cell lines, TJ-GBC2 cell line having a higher invasion ability and the highest migration ability compared to GBC-SD, NOZ, OCUG-1 and SGC-996, and positive expression of metastatic-related marker MMP9 and nm23 in the xenograft of TJ-GBC2 cells in nude mice, which is consistent with result of primary GBC, we thus identified TJ-GBC2 as a highly aggressive GBC cell line.