Abstract
Background/Aim: We and others have previously shown that cell fusion plays an important role in cancer metastasis. Color coding of cancer and stromal cells with spectrally-distinct fluorescent proteins is a powerful tool, as pioneered by our laboratory to detect cell fusion. We have previously reported color-coded cell fusion between cancer cells and stromal cells in metastatic sites by using color-coded EL4 murine lymphoma cells and host mice expressing spectrally-distinct fluorescent proteins. Cell fusion occurred between cancer cells or, between cancer cells and normal cells, such as macrophages, fibroblasts, and mesenchymal stem cells. In the present study, the aim was to morphologically classify the fusion-hybrid cells observed in the primary tumor and multiple metastases EL4 formed from cells expressing red fluorescent protein (RFP) in transgenic mice expressing green fluorescent protein (GFP), in a syngeneic model. Materials and Methods: RFP-expressing EL4 murine lymphoma cells were cultured in vitro. EL4-RFP cells were harvested and injected intraperitoneally into immunocompetent transgenic C57/BL6-GFP mice to establish a syngeneic model. Two weeks later, mice were sacrificed and each organ was harvested, cultured, and observed using confocal microscopy. Results: EL4 intraperitoneal tumors (primary) and metastases in the lung, liver, blood, and bone marrow were formed. All tumors were harvested and cultured. In all specimens, RFP-EL4 cells, GFP-stromal cells, and fused yellow-fluorescent hybrid cells were observed. The fused hybrid cells showed various morphologies. Immune cell-like round-shaped yellow-fluorescent fused cells had a tendency to decrease with time in liver metastases and circulating blood. In contrast fibroblast-like spindle-shaped yellow-fluorescent fused cells increased in the intraperitoneal primary tumor, lung metastases, and bone marrow. Conclusion: Cell fusion between EL4-RFP cells and GFP stromal cells occurred in primary tumors and all metastatic sites. The morphology of the fused hybrid cells varied in the primary and metastatic sites. The present results suggest that fused cancer and stromal hybrid cells of varying morphology may play an important role in cancer progression.
- EL4
- murine
- malignant lymphoma
- syngeneic mouse model
- red fluorescent protein
- green fluorescent protein
- color-coded imaging
- fused cells
- tumor-associated macrophage
- cancer-associated fibroblast
- metastasis
Our laboratory previously developed the technology to distinguish different cell types in vivo by transfecting them with spectrally-distinct fluorescent proteins. This technology could brightly distinguish cancer and stromal cells in vivo (1-15). Color-coded imaging with fluorescent proteins also visualized fusion between cancer cells and between cancer and normal cells, such as immune cells, mesenchymal stem cells (MSCs), and fibroblasts (14-16). Previous reports suggested that fusion products between cancer cells, or between cancer cells and stromal cells, had higher malignant potential than the parental cells (16-24), including increased metastasis (16, 18, 19, 25, 26).
In our previous report, we demonstrated EL4-red fluorescent protein (RFP)-expressing murine lymphoma cells implanted in the abdominal cavity of green fluorescent protein (GFP)-expressing-transgenic mice resulted in yellow-fluorescent fusion cells in the bone marrow and ascites (14). Similar results were found when mouse Colon 26 RFP cells were transplanted to the GFP transgenic mice (15).
In the present study, EL4-RFP cells were injected intraperitoneally into transgenic immunocompetent C57/BL6-GFP mice. The EL4-RFP cells formed intraperitoneal tumors and metastases in multiple organs. We harvested and cultured these tumors, and observed them with FV1000 confocal microscopy. We observed yellow-fluorescent hybrid fusion cells with different morphologies in the primary tumor and multiple metastases, suggesting that cell fusion between cancer cells and different types of stromal or other normal cells is part of the cancer-progression process.
Materials and Methods
Cell line and culture conditions. EL4-RFP cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), and 1% penicillin and streptomycin (GIBCO-BRL, Grand Island, NY, USA). The cells were cultured at 37°C in a 5% CO2 incubator.
GFP transgenic mice. Transgenic C57/BL6-GFP mice (27) were obtained from the Research Institute for Microbial Disease (Osaka University, Osaka, Japan). The C57/BL6-GFP mice express the Aequorea victoria GFP gene under the control of the chicken β-actin promoter and cytomegalovirus enhancer (27).
RFP fluorescent protein transduction of lymphoma cells. Murine EL4 lymphoma cells were labeled with RFP as previously reported using retroviruses expressing RFP (14).
Malignant lymphoma metastasis model. The C57/BL6-GFP transgenic mice were used as hosts. EL4-RFP lymphoma cells growing in vitro were first harvested and washed three times with cold serum-free medium, and then resuspended in serum-free RPMI 1640 medium. EL4-RFP cells (2.0×106) were injected into the abdominal cavity of C57/BL6-GFP transgenic mice. Two weeks later, the mice were sacrificed and the intraperitoneal tumor (primary tumor) and metastases were harvested. The cells from the primary tumor and metastatic tumors were cultured for several weeks.
Tumor imaging. The SZX7 microscope and FV1000 confocal microscope (Olympus Corp., Tokyo, Japan) were used for imaging (28-30).
Study approval. All experiments were conducted in accordance with the Institutional Guidelines of Gifu University and approved by the Animal Research Committee and the Committee on Living Modified Organisms of Gifu University (approval number AG-P-N-20230033).
Statistical analysis. All data were analyzed using the Fisher Exact Test. p-Values ≤0.05 are considered significant. Statistical analyses were performed with JMP pro ver 17.0.0 (SAS Institute, Cary, NC, USA).
Results and Discussion
RFP-expressing EL4 mouse malignant-lymphoma cells (Figure 1A) were injected intraperitoneally into immunocompetent C57/BL6-GFP transgenic mice. Two weeks later, EL4-RFP cells formed intraperitoneal tumors (primary tumor) and metastases were observed in the liver and lung. The primary tumor and metastases in the liver and lung were harvested and observed with the SZX7 microscope (Figure 1). Multiple metastatic lesions were observed in the liver in bright field, while lung metastases could only be observed with an RFP filter. The harvested intraperitoneal tumor, metastases, circulating blood and bone marrow were harvested and cultured for several weeks. The cultured cells from each tumor site were observed and images were captured with the Olympus FV1000 confocal microscope.
Images of EL4-RFP cells. A) EL4-RFP murine malignant lymphoma cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum. EL4-RFP cells growing in culture were observed with the Olympus FV1000 confocal microscope (Bar=50 μm). B) Bright-field and fluorescence images of EL4-RFP cells growing in the liver, intraperitoneal space, and lung 14 days after transplantation of the EL4-RFP cells to the abdominal cavity of transgenic GFP immunocompetent mice. Arrows indicate EL4 lymphoma metastatic tumors. Metastases in the lung could only be observed with an RFP filter. Images were captured with an SZX7 microscope (Bar=10 mm).
The EL4-RFP cells, stromal-GFP cells derived from the host mice, and various shaped EL4-RFP-mouse-GFP fused yellow-fluorescent cells were observed in the intraperitoneal tumor and all metastases (Figure 2). These cells were counted, and the ratio of fused cells to non-fused cells was calculated according to the tumor site on day-3, day-8, and day-13 of culture (Figure 3). The ratio of the fused yellow-fluorescent cells varied by culture time, and peak levels were calculated to be 2.4% in liver metastases, 0.2% in circulating blood, 1.5% in intraperitoneal tumors, 2.4% in lung metastases, and 1.3% in the bone marrow on and after day-13 (Figure 3). Although fused cells were a small proportion of all observed cells, fused yellow-fluorescent cells were observed in the primary tumor and all metastatic sites. Furthermore, a significant increase in the ratio of fused cells over time in culture was confirmed in the intraperitoneal tumor and lung metastases, and a tendency for an increased ratio of fused cells was also observed in the bone marrow. In the liver metastases and circulating blood, the ratio of fused cells tended to decrease over time in culture.
Fluorescence images of EL4-RFP cells fused with host GFP cells, cultured from each organ on the indicated day (Bar=50 μm). White arrows indicate round-shaped immune-cell-like fused hybrid cells. Red arrows indicate spindle-shaped fibroblast-like fused hybrid cells. Yellow arrows indicate dendritic-cell-like fused hybrid cells. EL4-RFP cells formed colonies with surrounding stromal cells in circulating blood. Images were captured with an Olympus FV1000 confocal microscope. (A) Liver metastasis, (B) circulating blood, (C) intraperitoneal tumor, (D) lung metastasis, (E) bone marrow.
Time course of the ratio of fusion hybrid cells from EL-4 tumors in organs cultured from transgenic GFP mice. The cell-culture time was divided into three periods (day 3, day 8 and, on and after day 13). A p-value ≤0.05 was considered a significant difference.
Subsequently, we classified fused cells into three categories morphologically. There were immune cell-like round-shaped fused yellow-fluorescent cells in the intraperitoneal tumor and all metastases, fibroblast-like spindle-shaped fused yellow-fluorescent cells in the intraperitoneal tumor and metastases except in the liver, and dendritic-shaped fused yellow-fluorescent cells in the intraperitoneal tumor and lung metastases (Figure 2). In every tumor site, either fibroblast-like yellow fluorescent fused hybrid cells or immune-like yellow fluorescent fused hybrid cells were observed along with EL4-RFP cells. The increase of fused cells in the intraperitoneal tumor and lung metastases was mainly due to an increase in fibroblast-like fused cells. In the cultured circulating blood, the ratio of fibroblast-like fused cells tended to increase even though the ratio of total fused cells decreased. At all tumor sites except liver metastases, the ratio of immune cell-like fused cells tended to decrease over time.
The morphologies of yellow-fluorescent fused cells depended on which type of stromal cell fused with EL4-RFP cells.
The fibroblast-like fused cells may have resulted from fusion of EL4-RFP cells with cancer-associated-fibroblasts (CAFs).
The macrophage-like fused cells may have resulted from fusion of EL4-RFP cells with GFP expressing tumor-associated macrophages (TAMs). A previous study showed that fused cells resulting between cancer cells and immune cells had increased migrative and invasive ability compared with parental cells. However, they exhibited decreased ability to proliferate (17). In the present study dendritic-cell like fused cells may have resulted from the fusion of EL4-RFP cells and tumor-associated dendritic cells.
Previously, we reported yellow-fluorescent hybrid fused cells between EL4-RFP cells and stromal-GFP cells in GFP transgenic mice. In this previous study, tumors formed in the GFP mice were subsequently implanted in the abdominal cavity of non-transgenic nude mice and they metastasized to the liver, perigastric lymph-node bone marrow, and ascites. Yellow-fluorescent fused cells were observed in the bone marrow and ascites only (14). In the present study, we implanted EL4-RFP tumors only in the immunocompetent GFP mouse, which is a syngeneic model. Yellow-fluorescent fused cells were observed in the primary tumor and all metastases, possibly due to the syngeneic immunocompetent host mouse. The hybrid fused cells showed various morphologies at each site and their ratios varied depending on the tumor locations and subsequent culture times.
Glinsky et al. and others have shown that fused cells between cancer cells or between cancer cells and stromal cells are more malignant than their parental cells (16-26). The pioneering in vivo imaging experiments of our laboratory using spectrally-distinct genetic fluorescent reporters made such experiments possible (1-15, 28-34).
In the present report, the ratio of immune cell-like fused cells were the majority of fused cells in the early period of culture of cells from the primary tumor and all metastatic cells. The ratio of fibroblast-like fused cells was higher in the late culture period than in the early period of culture except for cells cultured from liver metastases. Hybrid fusion cells may enhance metastasis and determine organ-specific metastasis. Future experiments will determine the in vivo behavior of each type of fused hybrid cell observed in the present study.
Footnotes
Authors’ Contributions
Koji Yamashita and Atsushi Suetsugu conceived and planned this 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. Robert M. Hoffman critically revised the manuscript. 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 9, 2024.
- Accepted June 10, 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).
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