Abstract
Background/Aim: Exosome exchange between cancer cells or between cancer and stromal cells is involved in cancer metastasis. We have previously developed in vivo color-coded labeling of cancer cells and stromal cells with spectrally-distinct fluorescent genetic reporters to demonstrate the role of exosomes in metastasis. In the present study, we studied exosome transfer between different pancreatic-cancer cell lines in vivo and in vitro and its potential role in metastasis. Materials and Methods: Human pancreatic-cancer cell lines AsPC-1 and MiaPaCa-2 were used in the present study. AsPC-1 cells contain a genetic exosome reporter gene labeled with green fluorescent protein (pCT-CD63-GFP) and MiaPaCa-2 cells express red fluorescent protein (RFP). Both cell lines were co-injected into the spleen of nude mice (n=5) to further study the role of exosome exchange in metastasis. Three weeks later mice were sacrificed and tumors at the primary and metastatic sites were cultured and observed by confocal fluorescence microscopy for exosome transfer. Results: The primary tumor formed in the spleen and metastasized to the liver, as observed macroscopically. Cells were cultured from the spleen, liver, lung, bone marrow and ascites. Transfer of exosomes from AsPC-1 to MiaPaCa-2 was demonstrated in the cultured cells by confocal fluorescence microscopy. Moreover, cell fusion was also observed along with exosome transfer. Exosome transfer did not occur during in vitro co-culture between the two pancreatic-cancer cell lines, suggesting a role of the tumor microenvironment (TME) in exosome transfer. Conclusion: The transfer of exosomes between different pancreatic-cancer cell lines was observed during primary-tumor and metastatic growth in nude mice. This cell-cell communication might be a trigger of cell fusion and promotion of cancer metastasis. Exosome transfer between the two pancreatic-cancer cell lines appears to be facilitated by the TME, as it did not occur during in vitro co-culture.
Exosomes are small (50-90 nm) extracellular vesicles (EVs) that contain large numbers of mRNAs, miRNAs and proteins, and transfer them to target cells (1, 2). Various types of cells, including cancer cells, produce exosomes. After secretion from donor cells, exosomes can be incorporated into target recipient cells. Although the functions of exosomes are incompletely understood, it is known that they play multiple roles in cell-cell communication, such as antigen presentation. Exosomes derived from cancer cells possess potential to enhance metastatic ability (3, 4). Additionally, it was reported that cancer cells show different organotropism to metastatic sites which may depend on the exosomes they secrete (5).
We developed in vivo color-coded labeling of cancer cells and stromal cells with spectrally-distinct genetic fluorescent reporters to demonstrate the role of exosomes in metastasis (6-10). In the present study we observed exosome transfer between different pancreatic-cancer cell lines in vivo, but not in vitro, and its potential role in metastasis.
We used the exosome reporter gene pCT-CD63-GFP transfected in cancer cells to observe exosome transfer in vivo. With the exosome reporter gene, we previously visualized in vivo exosome transfer from breast cancer cell lines to mouse lung tissue cells as well as exosomes released in the blood circulation (6). We have also observed exosomes derived from pancreatic cancer cells transferred to macrophages, lung tissue cells and bone marrow in vivo (7). We have also observed exosome transfer in vivo within a pancreatic cancer cell line and concluded it enhanced metastasis (8). These previous reports used spectrally-distinct genetic fluorescent reporters to visualize exosome-based cell-cell communication between cancer cells and multiple types of normal cells.
In the present report, we observed exosome transfer between two different types of pancreatic cancer cell lines in vivo and its effect on metastasis, but could not observe exosome transfer between the pancreatic-cancer cell lines when co-cultured in vitro.
Materials and Methods
Cell lines and culture conditions. MiaPaCa-2 human pancreatic adenocarcinoma cells, and AsPC-1 human pancreatic adenocarcinoma cells, were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin and streptomycin (GIBCO, Grand Island, NY, USA). The cells were cultured at 37°C in a 5% CO2 incubator. MiaPaCa-2 cells were engineered to stably express RFP as previously reported (9, 10).
Lentiviral exosome labeling. AsPC-1 cells were previously transduced with a lentiviral vector, containing the pCT-CD63-GFP gene (System Biosciences, Palo Alto, CA, USA), where the tetraspanin CD63 gene is fused to GFP for tracking exosomes (11).
Mice. Five 6-week-old BALB/c-nu/nu male mice (The Jackson Laboratory Japan, Inc., Yokohama, Japan) were used in the present study. Mice were housed in a barrier facility on a high-efficiency particulate arrestance (HEPA)-filtered rack under standard conditions of 12-h light/dark cycles. Mice were fed with an autoclaved laboratory rodent diet.
Pancreatic adenocarcinoma metastasis model. MiaPaCa-2 cells expressing RFP and AsPC-1 transduced with pCT-CD63-GFP were cultured in vitro and washed three times with cold serum-free medium, then resuspended in serum-free RPMI 1640 medium. MiaPaCa-2 cells expressing RFP (1.0×106) and AsPC-1 cells transduced with pCT-CD63-GFP (1.0×106) were co-injected into the spleen of mice. Three weeks later, the mice were sacrificed, and the splenic tumor (primary tumor) and metastases were harvested. The cells from primary tumor and metastatic tumors were cultured for two weeks (Figure 1A).
(A) Experimental schema. (B) Fluorescence image of AsPC-1 cells which contain the pCT-CD63-GFP-exosome reporter gene. Yellow arrows indicate GFP-exosomes. (C) Image of a MiaPaCa-2 cell expressing RFP in the cytoplasm. Images were captured with an Olympus FV1000 confocal microscope (Bar=50 μm). (D) Schematic diagram of pCT-CD63-GFP-expressing exosomes in an AsPC-1 cell. (E) Schematic diagram of a MiaPaCa-2-RFP cell. (F) Co-culture of AsPC-1/pCT-CD63-GFP cells and MiaPaCa-2 cells expressing RFP in vitro. GFP: Green fluorescent protein; RFP: red fluorescent protein; BM: bone marrow.
Tumor imaging. The SZX7 microscope and FV1000 confocal microscope (Olympus Corp. Tokyo, Japan) were used for imaging.
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.
Statistical analysis. All data were analyzed using the Fisher’s Exact Test. p-values ≤0.05 were considered significant.
Results
MiaPaCa-2 is an undifferentiated pancreatic adenocarcinoma cell line and was established from a primary tumor of a 65-year-old Caucasian man in 1977 (12). AsPC-1 is a pancreatic adenocarcinoma which ranged from well- to poorly-differentiated and was established from ascites of a 62-year-old Caucasian woman with adenocarcinoma of the head of the pancreas in 1982 (13). Although MiaPaCa-2 is less differentiated than AsPC-1, AsPC-1 was established from ascites and may possess greater stemness (14, 15).
MiaPaCa-2 cells expressing RFP (RFP-MiaPaCa-2) and AsPC-1 cells expressing pCT-CD63-GFP (AsPC-1/GFP-exosome) were co-cultured (Figure 1B and C). The transfer of exosomes from AsPC-1/GFP-exosome to RFP-MiaPaCa-2 was not observed in co-cultures of these cells in the same dish in vitro (Figure 1F).
RFP-MiaPaCa-2 cells (1.0×106) and AsPC-1/GFP-exosome cells (1.0×106) were co-injected into the spleen of five nude mice. Three weeks later, after confirmation of tumor formation in the spleen, mice were sacrificed. Primary splenic tumors and ascites were observed in all five mice and liver metastases were observed in four out of five mice. Lung metastases were not visible macroscopically (Figure 2). The resected splenic tumor and liver metastasis were observed ex vivo with the Olympus FV1000 confocal microscope and cell populations expressing RFP and GFP were identified (Figure 2). Subsequently, the splenic tumor, liver, lung, bone marrow and ascites were harvested from each mouse, and cultured in vitro.
Imaging of a resected spleen primary tumor and liver metastases. Images were captured with an Olympus FV1000 confocal microscope (Bar=50 μm). (A) liver metastasis, (B) splenic primary tumor. GFP: Green fluorescent protein; RFP: red fluorescent protein.
After 14 days of culture, cancer cells from each organ were imaged with a confocal microscope. Among the cells from the splenic tumor, liver metastasis and ascites, in addition to RFP-MiaPaCa-2 and AsPC-1/pCT-CD63-GFP cells, RFP-MiaPaCa-2-pCT-CD63-GFP cells were found to contain GFP-expressing exosomes derived from AsPC-1/pCT-CD63-GFP cells (Figure 3). This result indicates exosome transfer from AsPC-1/pCT-CD63-GFP to RFP-MiaPaCa-2 cells. In the cells from bone marrow and lung, although MiaPaCa-2 cells containing GFP-exosomes were not observed, there were exosome-containing stromal cells such as macrophages (Figure 4). Some of the RFP-MiaPaCa-2 cells with exosome transfer were multinucleated possibly due to cell fusion between RFP-MiaPaCa-2 and AsPC-1/pCT-CD63-GFP or other stromal cells which may be related to exosome transfer (Figure 5).
Fluorescence images of AsPC-1/pCT-CD63-GFP cells, MiaPaCa-2-RFP cells and stromal cells cultured from each organ. Solid white arrows indicate AsPC-1/pCT-CD63-GFP cells and dashed white arrows indicate pCT-CD63-GFP-exosomes incorporated into MiaPaCa-2-RFP cells. Images were captured with an Olympus FV1000 confocal microscope. (A) ascites, (B) splenic tumor, (C) liver metastasis (Bar=100 μm). GFP: Green fluorescent protein; RFP: red fluorescent protein.
Fluorescence images of AsPC-1/pCT-CD63-GFP cells, MiaPaCa-2-RFP cells and stromal cells cultured from each organ. White arrows indicate AsPC-1/pCT-CD63-GFP cells. Red arrow indicates a MiaPaCa-2-RFP cell. Yellow arrows indicate macrophages containing pCT-CD63-GFP-expressing exosomes. Images were captured with an Olympus FV1000 confocal microscope. (A) bone marrow, (B) lung (Bar=100 μm). GFP: Green fluorescent protein; RFP: red fluorescent protein; BM: bone marrow.
Fluorescence images of cells from splenic tumors and liver metastases. Yellow arrows indicate multinucleated MiaPaCa-2-RFP cells containing pCT-CD63-GFP-expressing-exosomes derived from AsPC-1/pCT-CD63-GFP cells. (A) splenic tumor, (B) liver metastasis (Bar=50 μm), (C) Schematic diagram of multinucleated MiaPaCa-2-RFP cell containing pCT-CD63-GFP-expressing exosomes. GFP: Green fluorescent protein. RFP: Red fluorescent protein.
In the present report, we demonstrated the transfer of exosomes between different pancreatic-cancer cell lines with RFP- GFP color-coded imaging. This result suggests that heterogeneous cancer cells can communicate with each other through exosomes, and the exchange might contribute to increased malignant potential of heterogeneous cancer cells. The ratio of exosome transfer in ascites was significantly higher than in liver metastases (p=0.000246) (Figure 6). This result suggests that sites farther distant from the primary site may possess a higher ratio of exosome transfer.
Ratios of MiaPaCa-2-RFP cells (RFP), AsPC-1/pCT-CD63-GFP cells (GFP) and MiaPaCa-2-RFP cells with transferred pCT-CD63-GFP-expressing exosomes (merge) in the primary tumor and metastatic organs. RFP: Red fluorescent protein; GFP: green fluorescent protein; BM: bone marrow. The numbers in the bar graphs indicate the percentage of each cell-type present in the organ.
Discussion
We previously reported exosome transfer between different types of cells (6-8) suggesting exosome transfer enhanced metastasis. These previous reports demonstrated the transfer of exosomes between the same type of cancer cells or from cancer cells to stromal cells, such as cancer-associated fibroblasts (CAFs) and macrophages (6-8). Cancer-derived exosomes have been reported to promote metastasis by developing a pre-metastatic niche of stromal cells (16, 17), or causing immunosuppression (18, 19).
Multinucleated cells with exosome transfer were observed in the present study (Figure 5). The multinucleated cells indicate possible exosome transfer as well as cell fusion which may have occurred simultaneously or consecutively. Fusion between two or more cells may have occurred and the fused cells may have increased metastatic potential compared to the parental cells (20-22).
In the present study, we also observed that exosome transfer between AsPC-1/pCT-CD63-GFP and MiaPaCa-2 cells did not occur during in vitro co-culture suggesting a critical role of the TME in exosome transfer.
Footnotes
Authors’ Contributions
Koji Yamashita and Atsushi Suetsugu conceived and planned the present study. Koji Yamashita and Atsushi Suetsugu carried out the experiments. Koji Yamashita, Atsushi Suetsugu, and Robert M. Hoffman contributed to the interpretation of the results. All Authors provided critical feedback and helped shape the research and manuscript.
Conflicts of Interest
None of the Authors have any conflicts of interest with regard to this study.
- Received May 8, 2024.
- Revision received June 7, 2024.
- Accepted June 12, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
