Abstract
Background/Aim: The interaction between pancreatic-cancer cells and stromal cells in the tumor microenvironment (TME) is of particular importance in cancer progression and metastasis. The present report demonstrates the role of cancer-associated fibroblasts (CAFs) and multinucleate pancreatic-cancer cells in peritoneal metastasis. Materials and Methods: An orthotopic mouse model of pancreatic cancer was established with the human pancreatic cancer cell line BxPC3, which stably expresses green fluorescent protein (GFP). Results: BxPC3-GFP cells formed peritoneal metastases by week 18 after orthotopic implantation. Using an Olympus FV1000 confocal microscope, multi-nucleated cancer cells were frequently observed in the peritoneal metastases. The primary pancreatic tumor and peritoneal-metastases were harvested, cultured and then transplanted subcutaneously. Subcutaneous tumors established from peritoneal-metastatic cells were larger than subcutaneous tumors established from primary-tumor cells. Subcutaneous tumors of each type were subsequently cultured in vitro. CAFs were observed growing out from the tumors established from peritoneal-metastatic cells, but not the tumors established from the primary cancer. Conclusion: The results of the present study suggest that multi-nucleated cancer cells and CAFs were related to peritoneal metastasis of pancreatic cancer.
- Pancreatic cancer
- nude mice
- orthotopic
- peritoneal metastasis
- cancer-associated fibroblasts
- green fluorescent protein
- imaging
Pancreatic cancer is characterized by a thick desmoplastic stromal matrix comprising abundant fibroblasts and immune cells that play an important role in cancer progression, metastasis, immunosuppression and resistance to chemotherapy (1, 2).
We previously reported color-coded imaging of cell dynamics in the tumor microenvironment (TME) including the interaction between cancer cells and stromal cells (3-5). The tumors acquired brightly-fluorescent stromal cells from transgenic host mice expressing a different color fluorescent protein from the cancer cells in the tumor. The stromal cells were stably associated with the tumors through multiple passages. The colored fluorescent protein-expressing stromal cells included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) (3).
Liver metastases and disseminated peritoneal metastases maintained the fluorescent-protein-expressing stroma from the primary tumor and possibly recruited additional fluorescent protein-expressing stroma, resulting in their very bright fluorescence. The fluorescent protein-expressing stroma included CAFs and TAMs in both the primary and metastatic tumors (6).
With high-resolution intravital imaging afforded by the Olympus FV1000 confocal microscope, the interaction of pancreatic cancer cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm and RFP-expressing pancreatic stellate cells could be clearly imaged in the liver and other metastases, suggesting that stellate cells participate in metastasis formation (5).
In the present study, we developed a nude-mouse orthotopic model of pancreatic cancer (7-12) resulting in peritoneal metastasis with the human pancreatic cancer cell line (BxPC3), which stably expresses GFP (BxPC3-GFP), in order to further understand the interaction of pancreatic cancer cells and stromal cells in the TME.
(A) Schematic representation of pancreatic cancer orthotopic xenograft. 35 days after subcutaneous injection of BxPC3-GFP cells, subcutaneous tumors were established. Subcutaneous tumors were harvested, minced into fragments and orthotopically transplanted into the pancreas tail of nude mice. 18 weeks after tumor transplantation, peritoneal metastasis lesions were observed. Black arrows indicate BxPC3-GFP tumors. (B) Bright-field and fluorescence image of the primary tumor and peritoneal metastases. Red arrows indicate peritoneal metastases. Blue arrow indicates the primary pancreatic tumor. Yellow arrow indicates gallbladder. (C) High-magnification fluorescence image of BxPC3-GFP tumors. Left panel shows a subcutaneous tumor. Middle panel shows a primary orthotopic pancreatic tumor. Right panel shows a peritoneal metastasis. Red arrows indicate mono-nuclear cancer cells. Yellow arrows indicate multi-nucleate cancer cells (Bar=20 μm). (D) Hematoxylin and eosin (H&E) stained section of BxPC3-GFP tumors. Left panel shows the primary orthotopic pancreatic tumor tissue. Right panel shows the peritoneal metastasis. Red arrows indicate multi-nucleated cells. (E) Five randomly-selected, low-magnification visual fields were quantified for the number of mono-nuclear and multi-nucleate cancer cells. The frequency of each cell type in the primary orthotopic pancreatic tumor and peritoneal metastases are plotted in the bar graphs (p=0.0003).
Materials and Methods
Cell line and culture condition. The human pancreatic cancer cell line BxPC3 was engineered to stably express green fluorescent protein (GFP) (BxPC3-GFP) (13-15). The cells were maintained in RPMI 1640 medium (Gibco-BRL, Grand island, NY, USA). supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco-BRL) and 1% penicillin and streptomycin (Gibco-BRL). The cell line was cultured in a humidified atmosphere containing 5% CO2 at 37°C.
Pancreatic cancer orthotopic BxPC3-GFP model. Six-week-old nude mice were used as the host. BxPC3-GFP cells were harvested by trypsinization and washed three times with cold serum-free medium, then re-suspended in serum-free RPMI 1640 medium. BxPC3-GFP cells (2.0×106) were then injected subcutaneously in 3 nude mice and formed tumors by day 35 after injection. The subcutaneous tumors were harvested and divided into 3 mm3 fragments and orthotopically transplanted to the pancreas tail of BALB/c nude mice, using 5-0 nylon surgical sutures (7-12). Metastases were observed 18 weeks after transplantation (Figure 1A). All surgical procedures were performed with the animals anesthetized by subcutaneous injection of 100 mg/kg ketamine.
Subcutaneous injection of primary pancreatic tumors and peritoneal metastasis. The primary pancreatic tumor and peritoneal metastases were harvested and the cells were cultured. Pancreatic cancer cells (2.0×106) from each source were injected subcutaneously in the right- and left-side back flank, respectively, of BALB/c nude mice. Thirty five days after injection, cells from each source formed subcutaneous tumors.
Tumor imaging. The SZX7 microscope and FV1000 confocal microscope, both from Olympus Corp. (Tokyo, Japan) and Dino-lite digital fluorescence microscope (AM4113T-GFBW Dino-Lite Premier; AnMo Electronics Corp, Hsinchu, Taiwan) were used for intravital and ex vivo imaging. The BX53 microscope (Olympus Corp) was used for histological sections.
Histology. Tumors were prepared for histological analysis using hematoxylin and eosin (H&E) staining using previously published protocols (16).
Statistical analysis. A two-sided t-test was used to determine statistical significance. A p-value of ≤0.05 was considered significant.
Study approval. All experiments were conducted in accordance with the Institutional Guidelines of Gifu University and were approved by the Animal Research Committee and the Committee on Living Modified Organisms of Gifu University (approval number 26-37).
Results and Discussion
Establishment of the pancreatic cancer orthotopic model. Peritoneal metastasis was observed 18 weeks after transplantation (Figure 1B). The primary pancreatic tumors and peritoneal metastases were clearly observed in both bright field and by fluorescence microscopy (Figure 1B, C). Both the primary tumor and peritoneal metastasis brightly expressed GFP. Peritoneal metastasis had multiple foci.
Cell types cultured from the original subcutaneous tumor, primary orthotopic tumor and peritoneal metastasis. Abnormal multinucleate cancer cells with a large cytoplasm were observed growing from the peritoneal metastases (Figure 1D). These abnormal cancer cells were not observed among the original BxPC3-GFP cells cultured before subcutaneous injection in mice and at a lower amount among cells cultured from the primary orthotopic tumor compared to cells grown from the peritoneal metastases (p=0.0003) (Figure 1E). Multi-nucleate cancer cells were also observed in a metastasis of a colon cancer cell line (17). These multi-nucleated cells may have resulted from cell fusion or other means by which 2 or more cells can recombine their genetic material and may contribute to greater potential of tumor progression and metastasis (18).
(A) Cells cultured from an orthotopic tumor (Figure 1A, No 1) and peritoneal metastasis (No 2), were implanted subcutaneously. The comparison of tumor size of each type and weight after 35 days are shown to the bar graph. (B) Cells cultured from the subcutaneous tumor derived from the primary pancreatic tumor or from a subcutaneous tumor derived from a peritoneal metastasis. Upper panels show cells growing from subcutaneous tumors derived from the primary pancreatic tumor. Lower panels show cells cultured from the subcutaneous tumor derived from the peritoneal metastasis. Left panels show GFP fluorescence images. Middle panels show bright field images. Right panels show merged images. Red arrows indicate cancer-associated fibroblasts (CAFs). CAFs were only observed growing from the tumor that originated from cells in the peritoneal metastasis (Bar=100 μm).
Tumorigenic potential of cells growing out from subcutaneous and the peritoneal metastasis. Cells that grew from subcutaneous tumor and peritoneal metastasis were separately implanted subcutaneously. Tumors that grew from cells derived from the peritoneal metastasis were significantly larger than cells derived from the subcutaneous tumor (Figure 2A).
CAFs found in the peritoneal metastasis. The excised primary pancreatic tumor cells and peritoneal metastasis were cultured for 18 weeks. Large numbers of fibroblasts were observed among the cells growing from the peritoneal metastasis cells but not the primary orthotopic tumor cells (Figure 2B). Therefore, these CAFs appeared to be related to tumor progression and metastasis (19, 20).
Conclusion
BxPC3-GFP cells formed peritoneal metastasis by week 18 after orthotopic implantation. Using an Olympus FV1000 confocal microscope, multi-nucleate cancer cells were observed frequently in the peritoneal metastasis. The primary pancreatic tumor and peritoneal-metastasis were harvested and cultured and each transplanted subcutaneously. Subcutaneous tumors established from peritoneal metastatic cells were larger than subcutaneous tumors established from the primary tumor cells. Subcutaneous tumors of each type were then cultured in vitro. CAFs and multi-nucleate cancer cells, were observed growing out from the tumors established from peritoneal metastatic cells and to a lesser extent from tumors established from the primary cancer. The results of the present study suggest that multi-nucleated cancer cells and CAFs were related to peritoneal metastasis of pancreatic cancer. Multi-nucleate cancer cells may arise by recombination with other cell types (18, 21).
Footnotes
This article is freely accessible online.
Conflicts of Interest
None of the Authors have any conflict of interest in regard to this study.
- Received April 25, 2017.
- Revision received May 24, 2017.
- Accepted May 25, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
