Abstract
Background/Aim: Our laboratory pioneered color-coded imaging of the tumor microenvironment (TME). We observed recruitment of cancer and stromal cells to the TME and recombination between cancer and stromal cells. The aim of the present study was to observe the dynamics of the TME by color-coded imaging during metastasis and in the formation of a pre-metastatic niche. Materials and Methods: Red-fluorescent protein (RFP-expressing) mouse colon-cancer 26 cells were initially injected subcutaneously in green-fluorescent protein (GFP) nude mice. The resulting subcutaneous tumors were harvested and cultured. The cultured subcutaneous tumors contained RFP colon cancer cells, GFP stromal cells and recombinant cancer-stromal cells expressing yellow fluorescence. After 14 days culture, the cells were injected into the spleen. Results: After splenic injection, colon-cancer 26 metastases were observed in the liver, ascites, and bone marrow. Using the Olympus FV1000 confocal microscope, the cells cultured from tumors and metastasis in each site were visualized. RFP colon-cancer cells, GFP stromal cells derived from host GFP nude mice, and recombinant yellow-fluorescent cells were observed in each organ. In addition, in the liver, areas with only GFP stromal cells were observed and assumed to be a pre-metastatic niche. Conclusion: Color-coded imaging demonstrated the dynamics of colon cancer and stromal cells at different metastatic sites including the formation of recombinant cancer-stromal cells.
- Colon 26
- colorectal cancer
- RFP
- GFP mouse
- recombinant cell
- tumor-associated macrophage
- cancer associated fibroblast
- metastasis
- red fluorescent protein
- color coded imaging
The tumor microenvironment (TME) is critical for tumor progression and response to therapy (1-2). We pioneered color-coded imaging with multi-spectral fluorescent protein to visualize the dynamics of the TME (3-16).
In a previous report, we established a syngeneic model of metastatic lymphoma using EL4 cells expressing red fluorescent protein (RFP). We demonstrated that abundant stromal cells were recruited to the metastatic sites (17, 18).
In a subsequent study with the EL4-RFP malignant lymphoma metastasis model, we identified yellow-fluorescent genetically-recombinant cells formed during metastasis from EL4-RFP cells and GFP stromal cells from the host GFP transgenic mouse (19). The recombinant yellow-fluorescent cells were observed only in ascites and bone marrow.
Glinsky et al. demonstrated that recombinant yellow-fluorescent metastasis precursor cells resulted from interaction of high-metastatic and low-metastatic prostate cancer cells, each expressing a different color fluorescent protein (20). The recombinant cancer cells were more metastatic than either parent.
In the present study, RFP colon cancer cells and GFP stromal cells derived from a subcutaneous tumor site were cultured, injected into the spleen of non-colored nude mice and formed liver metastasis, bone metastasis, and ascites. We observed and quantified cancer cells, stromal cells, and recombinant cancer-stromal cells at each site and also observed pre-metastatic niche formation.
Materials and Methods
Cell line and culture condition. Colon 26, a mouse colon cancer cell line, was engineered to express red fluorescent protein (RFP) (22-23). The cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin and streptomycin (Gibco-BRL, Grand Island, NY, USA). The cells were cultured at 37°C in a 5% CO2 incubator.
GFP transgenic nude mice. Transgenic C57/B6-GFP mice (24) were obtained from the Research Institute for Microbial Diseases (Osaka University, Osaka, Japan). The C57/B6-GFP mice expressed the Aequorea Victoria GFP under the control of the chicken β-actin promoter and cytomegalovirus enhancer. The GFP mouse was crossed with non-transgenic nude mice to establish GFP-transgenic nude mice (25-26).
Colon 26-RFP subcutaneous tumor. Colon 26 RFP cells were first harvested from culture by trypsinization and washed three times with cold serum-free medium. Colon 26 RFP cells (2.0×106) were injected subcutaneously in GFP transgenic nude mice. Subsequent tumors were harvested by resection after 2 weeks growth. The tumor was minced into 1 mm3 fragments and cultured for 14 days (Figure 1A).
Colon 26 colon cancer liver metastasis model formation. The cells harvested from the culture of the subcutaneous colon 26-RFP-GFP tumor were injected into the spleen of 6-week-old Balb/c non-transgenic nude male mice. After 2 weeks, the mice were sacrificed and explored for primary tumor growth and metastases (Figure 2A).
Tumor imaging. The SZX7 microscope, and the FV1000 confocal microscope, both from Olympus Corp. (Tokyo, Japan), were used for intravital and ex vivo imaging.
Study approval. All experiments were conducted in accordance with the institutional guidelines of the 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
Color-coded imaging of the TME of subcutaneous tumor. Colon 26 RFP cells were injected subcutaneously in GFP nude mice. The resulting subcutaneous tumor was harvested (Figure 1A) and consisted of RFP colon cancer cells, GFP stromal cells derived from GFP nude mice, and a few recombinant yellow-fluorescent cancer-stromal cells (Figure 1B). The colon 26 RFP-GFP subcutaneous tumors were minced, cultured and observed in vitro for 14 days. The RFP colon cancer cells, GFP stromal cells, and various shaped recombinant yellow-fluorescent cancer stromal cells were observed. The recombinant yellow-fluorescent cells had one or more nuclei. These cells show that colon cancer cells have the ability to recombine with stromal cells (Figure 1C-E).
Color-coded imaging of the TME during metastasis. In the next stage, a colon cancer metastasis model was established using cells cultured from colon 26 RFP-GFP subcutaneous tumors. The color-coded cells cultured from the subcutaneous tumor were injected in the spleen of non-colored nude mice (Figure 2A). Colon 26 cancer metastases were observed in the liver, ascites, and bone marrow, as well as a primary tumor was formed at the spleen. Subsequent metastases were formed in the liver, which comprised colon-26 cancer cells, GFP-expressing stromal cells, and yellow fluorescent recombinant cancer-stromal cells (Figures 2B and 3A). In contrast, in another area of the liver only GFP stromal cells were observed and no cancer cells were found (Figure 2B). At this point, RFP colon cancer cells could not be identified. However, on day 15, a few RFP colon cancer cells appeared and had increased (Figure 3B).
Distribution of cancer, stromal, and recombinant cancer-stromal cells.
In recent years, it has been proposed that the pre-metastatic niches are formed before the arrival of cancer cells (27-28). This supportive microenvironment of metastatic niche is thought to allow cancer cells to colonize and grow. The high-magnification images in Figure 3B possibly catch this pre-metastatic niche. The GFP stromal cells in this area of the liver comprised macrophages (TAM), immune cells, endothelial cells, and fibroblasts (CAF).
In the spleen, the primary cancer-cell injection site, a primary tumor was formed. In the high-magnification images, RFP colon cancer cells and GFP stromal cells derived from host GFP nude mice were observed ex vivo (Figure 2C). In the high-magnification images of cells cultured from the spleen, ascites, and bone marrow, RFP colon cancer cells, GFP stromal cells, and recombinant yellow-fluorescent cancer-stroma cells were observed. The yellow fluorescent cells had various morphologies such as dendritic cells, macrophages, and fibroblasts (Figure 4A-C).
To compare the primary and metastatic tumors, the number of colon 26 RFP cells, GFP stromal cells, and recombinant yellow-fluorescent cancer-stroma cells in the tumors were counted from each organ (Table I). Recombinant yellow-fluorescent cancer-stroma cells were observed in the primary spleen tumor and all metastatic sites.
Future studies will focus on the properties of the recombinant yellow fluorescent cancer stroma cells and how they influence the metastatic behavior of the tumor as well as the dynamics of the metastatic niches all made possible by color-coded imaging.
Subcutaneous colon 26 RFP-GFP tumors. A: Experimental scheme. B: High-magnification images of the harvested colon 26 RFP-GFP subcutaneous tumors were captured with the Olympus FV1000 confocal microscope. Upper panels: RFP colon cancer cells and GFP stromal cells derived from host GFP nude mice were observed (Bar=50 μm). Lower panels: Blue arrows indicate recombinant yellow-fluorescent cells (Bar=30 μm). C: High-magnification images of cells cultured from harvested colon 26 RFP tumors on day 3. Upper panels: Some recombinant yellow-fluorescent cells were observed (Bar=50 μm). Lower panels: Round recombinant yellow-fluorescent cells were observed (Bar=30 μm). D: High-magnification images of cells cultured from harvested colon 26 RFP tumors on day 7. Upper panels: RFP colon 26 cancer cells and GFP stromal cells derived from host GFP nude mice were observed (Bar=50 μm). Lower panels: White arrows indicate recombinant yellow-fluorescent cells that had three nuclei. (Bar=30 μm). E: High-magnification images of cells cultured from harvested colon 26 RFP tumors on day 14. Upper panels: White arrows indicate spindle-shaped yellow-fluorescent recombinant cells (Bar=20 μm). Lower panels: Yellow arrows indicate a recombinant yellow-fluorescent cell that has two nuclei. B-E: All images were captured with the Olympus FV1000 confocal microscope.
Colon 26 RFP-GFP tumors were formed in the liver of non-colored nude mice. A: Experimental schema. B: Left panels: Liver-metastasis images were obtained with the SZX7 microscope (Bar=10 mm). Yellow arrows (1) indicate liver-metastases area. Blue arrows (2) indicate the area that contained only GFP-expressing stromal cells without apparent cancer cells. Right upper panels: High-magnification images of liver metastases were obtained with the Olympus FV1000 confocal microscope. Both RFP colon-cancer cells and GFP stromal cells derived from host GFP nude mice were observed (Bar=50 μm). Right lower panels: High-magnification images were obtained with the Olympus FV1000 confocal microscope. Only GFP stromal cells derived from host GFP nude mice were observed (Bar=100 μm). C: Left panels: The tumor was formed in the spleen (primary injection site). The image was obtained with the SZX7 microscope (Bar=10 mm). Red arrows indicate tumor area. Right panels: High-magnification images of splenic tumor were captured with the Olympus FV1000 confocal microscope. Many RFP colon-cancer cells and a few GFP stromal cells derived from host GFP nude mice were observed. (Upper panels: Bar=100 μm; Lower panels: Bar=50 μm).
High-magnification images of cells cultured from liver metastases. A: High-magnification images of cells cultured from liver metastases. Left upper panels: White arrows indicate a dendritic-shaped recombinant yellow-fluorescent cancer-stromal cell. (Bar=30 μm). Left middle panels: Yellow arrows indicate round-shaped recombinant yellow-fluorescent cancer-stromal cells (Bar=20 μm). Left lower panels: A mass formed from RFP cancer cells and GFP stromal cells was observed. (Bar=50 μm). Right upper and middle panels: Blue arrows indicate a recombinant yellow-fluorescent cancer-stromal cell (Bar=30 μm). Right lower panels: RFP colon-cancer cells surround GFP stromal cells cultured from the tumor (Bar=50 μm). B: High-magnification images of cells cultured from liver tissues expressing only GFP. Left upper panels: Red arrows indicate a fibroblast. White arrows indicate a macrophage. No RFP colon-cancer cells were observed (Bar=30 μm). Left middle panels: Yellow arrows indicate a few RFP colon-cancer cells (Bar=50 μm). Left lower panels: Blue arrows indicate a cell that has RFP cytoplasm and GFP nuclei. (Bar=20 μm). Right panels: An increase of RFP colon-cancer cells (white arrows indicate) was observed (Bar=50 μm). A-B: All images were captured with the Olympus FV1000 confocal microscope.
High-magnification images of cells cultured from splenic tumors, ascites and bone marrow using confocal microscopy. A: High-magnification images of cells cultured from splenic tumor. Left upper panels: Yellow arrows indicate recombinant yellow-fluorescent cancer-stromal cells (Bar=50 μm). Left lower panels: Blue arrows indicate a recombinant yellow-fluorescent cancer-stromal cell (Bar=50 μm). Right panels: Colon 26-RFP cancer cells and GFP stromal cells in culture. (Bar=100 μm). B: High-magnification images of cells cultured from ascites. Left upper panels: Many colon 26-RFP cells and few GFP stromal cells were observed (Bar=100 μm). Left lower panels: White arrows indicate recombinant yellow-fluorescent cancer-stromal cells (Bar=30 μm). Right panels: Blue arrows indicate various shaped recombinant yellow-fluorescent cancer-stromal cells (Bar=40 μm). C: High-magnification images of cells cultured from bone marrow. Left panels: Colon 26-RFP cancer cells and GFP stromal cells were observed (Bar=30 μm). Right panels: White arrows indicate recombinant yellow-fluorescent cancer-stromal cells. A-C: All images were captured with the Olympus FV1000 confocal microscope.
Footnotes
This article is freely accessible online.
- Received June 4, 2018.
- Revision received June 17, 2018.
- Accepted June 18, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
