Imaging the role of the tumor microenvironment in tumor progression and metastasis

Our laboratory pioneered the use of fluorescent proteins for in vivo imaging. These early experiments used green fluorescent protein to image tumor growth and metastasis [1-6] . Subsequently cancer cells and stromal cells were color-coded with different colored fluorescent proteins which began the field of fluorescence imaging of the tumor microenvironment (TME) [3, 7-13] , and demonstrated the essential role of tumor-associated host cells in tumor progression and metastasis [14, 15] . Color-coded imaging of the TME has also enabled important discoveries of its cellular components [16,


Color-coded imaging of the tumor microenvironment (TME)
Our laboratory pioneered the use of fluorescent proteins for in vivo imaging.These early experiments used green fluorescent protein to image tumor growth and metastasis [1][2][3][4][5][6] .Subsequently cancer cells and stromal cells were color-coded with different colored fluorescent proteins which began the field of fluorescence imaging of the tumor microenvironment (TME) [3,[7][8][9][10][11][12][13] , and demonstrated the essential role of tumor-associated host cells in tumor progression and metastasis [14,15] .Color-coded imaging of the TME has also enabled important discoveries of its cellular components [16,17] .

Imaging recruitment of stromal cells to the TME
A model of recruitment of the TME was established by implanting non-colored HCT-116 human colon cancer cells in the spleen of transgenic GFP nude mice.The HCT-116 cells formed liver metastasis.The GFP nude mice express GFP in essentially all organs and tissues but GFP expression in the parenchymal cells of the liver is minimal.The HCT-116 cells formed tumor and recruited bright GFP-expressing non-parenchymal cells to the liver metastasis, which were readily imaged by their strong contrast of bright GFP compared to the dim GFP fluorescence of the liver parenchymal cells.The recruited stromal cells in the tumor expressed desmin indicated that they were cancer-associated fibroblasts (CAFs) which may have been necessary to promote growth of the liver metastasis [18] .

Color-coded imaging of the TME
The color-coded fluorescence imaging was effected by using red fluorescent protein (RFP)-expressing tumors growing in GFP-expressing transgenic mice.This model showed the details of the tumor-stroma interaction, especially tumor-induced angiogenesis and tumor-infiltrating lymphocytes.The GFP-expressing tumor vasculature, both nascent and mature, could be readily distinguished interacting with the RFP-expressing cancer cells.GFP-expressing dendritic cells were visualized contacting

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RFP-expressing cancer cells with their dendrites.GFP-expressing macrophages were observed engulfing RFP-expressing cancer cells.GFP lymphocytes were seen surrounding cells of the RFP tumor.Color-coded fluorescence imaging visualized the different cellular elements of the TME [7] .

Stromal cells in the TME are essential for metastasis
Dual color HCT-116 colon cancer cells expressing GFP in the nucleus nad RFP in the cytoplasm were injected into the spleen or portal vein (PV) of GFP transgenic nude mice.The HCT-116-GFP-RFP cells formed tumors in the liver, which were surrounded by a ring of GFP-expressing spleen cells (Figure 1).The question was what is the role of the spleen cells in the formation of the metastases?This was answered in the following experiment: The HCT-116-GFP-RFP cells were injected in the portal vein and did not form metastases in the liver as they did when injected in the spleen.However, when the HCT-116-GFP-RFP cells were co-injected with splenocytes in the portal vein, tumors formed in the liver, thereby demonstrating that the splenic cells were necessary for metastasis of HCT-116-GFP-RFP cells [14] .
In another tumor model to determine the role of splenocytes on tumor growth and metastasis, dual color human XPA-1 pancreatic cancer cells expressing GFP in the nucleus and RFP in the cytoplasm were implanted orthotopically in the pancreas of nude mice.After the tumor was established, GFP or RFP expressing splenocytes were isolated from immunocompetent GFP or RFP transgenic mice.At first, the GFP-or RFP-expressing splenocytes were found in the lung, liver, spleen and pancreas after i.v.injection.However, after 4 days, GFP-or RFP-expressing splenocytes were surrounding the orthotopic tumor in the  B. in contrast to the PV where the cells rapidly died, when the same cells were injected into the spleen, multiple metastatic tumors were imaged in the liver in 8 of 15 mice.Liver micrometastases were imaged by day 7.By day 30, large tumor colonies were imaged in the liver and spleen.C. by day 60, large tumors were imaged in the liver [14] .D. Sister slides analyzed by florescence and Immunohistochemistry. Left panel: frozen section from a liver metastasis showing by fluorescence microscopy GFP splenocytes surrounding the HCT-116-RFP colon cancer cells.Right panel: immunohisto-chemical analysis shows that the GFP-host cells stained positive (+) with monoclonal antibodies to CD11c confirming that these cells were mouse splenocytes.
pancreas, as well as locating in the lymph nodes and the spleen.These results indicate that the cancer recruited the splenocytes in order to progress, further indicating the role of splenocytes in tumor development [15] .Liver tumors were also imaged to recruit stroma.After splenic injection of non-colored Huh-7 human hepatoma cells in RFP transgenic nude mice, they began to form tumors in the nude mouse liver.The non-colored growing liver tumors recruited RFP-expressing stromal cells and RFP-expressing blood vessels to the tumor, visualized with fluorescence imaging.The stromal cells recruited by the liver tumors expressed desmin suggested they were CAFs.These results indicated that the recruited CAFs and blood vessels promoted the growth of the liver tumors [19] .

Conclusions
The TME is essential for tumor progression including metastasis.Color-coded imaging of the TME has enabled the identification of non-cancer TME cell types that interact with cancer cells and effect tumor progression and metastasis.Color-coded imaging of the TME will lead to new visual targets for cancer therapy, including critical stromal cells as well as cancer cells.

Figure 1 .
Figure 1. A. schematic representation of experimental protocol.Human HCT-116-RFP-GFP colon cancer cells (2.0 × 10 6 /50 µL Matrigel) were injected in the PV or spleen of transgenic GFP nude mice during open laparotomy.B. in contrast to the PV where the cells rapidly died, when the same cells were injected into the spleen, multiple metastatic tumors were imaged in the liver in 8 of 15 mice.Liver micrometastases were imaged by day 7.By day 30, large tumor colonies were imaged in the liver and spleen.C. by day 60, large tumors were imaged in the liver[14] .D. Sister slides analyzed by florescence and Immunohistochemistry. Left panel: frozen section from a liver metastasis showing by fluorescence microscopy GFP splenocytes surrounding the HCT-116-RFP colon cancer cells.Right panel: immunohisto-chemical analysis shows that the GFP-host cells stained positive (+) with monoclonal antibodies to CD11c confirming that these cells were mouse splenocytes.