Open Access

The Role of Microsurgery and Fluorescent-reporter Genes in Establishing Mouse Models for Real-Time Imaging of Metastatic Cancer-Cell Trafficking and Colony Formation: A Revolutionary and Disruptive Technology for Metastasis Research

MORINAGA SEI 1 2 3
YAMAMOTO NORIO 3
YAMAUCHI KENSUKE 3
HAYASHI KATSUHIRO 3
KIMURA HIROAKI 3
MIWA SHINJI 3
IGARASHI KENTARO 3
HIGUCHI TAKASHI 3
TSUCHIYA HIROYUKI 3
DEMURA SATORU 3
  &  
HOFFMAN ROBERT M. 1 2

1AntiCancer Inc., San Diego, CA, U.S.A.

2Department of Surgery, University of California, San Diego, CA, U.S.A.

3Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan

Cancer Diagnosis & Prognosis Sep-Oct; 4(5): 544-557 DOI: 10.21873/cdp.10362
Received 21 May 2024 | Revised 03 October 2024 | Accepted 19 June 2024
Corresponding author
Robert M. Hoffman, Ph.D., AntiCancer Inc, 7917 Ostrow St, Suite B, San Diego, CA, 92111, U.S.A. Tel: +1 6198852284, email: all@anticancer.com

Abstract

The field of experimental microsurgery was pioneered by the great microsurgeon Sun Lee, who developed the foundation of transplant surgery in the clinic. Dr Lee also played a seminal role in introducing microsurgery to establish mouse models of cancer. In 1990, at the age of 70, Dr Lee demonstrated microsurgery techniques to the mouse-model team at AntiCancer Inc., leading to the development of the surgical orthotopic implant (SOI) technique and the first orthotopic mouse models of cancer that metastasized in a pattern similar to clinical cancer. At the beginning of the present century, one of us (NY) from Kanazawa University School of Medicine became a visiting scientist at AntiCancer to learn SOI and develop mouse models of cancer using cancer cells expressing fluorescent reporter genes, such as green fluorescent protein (GFP) and red fluorescent protein (RFP), in order to image metastatic cancer cells trafficking in real time. Since then, a total of eight young surgeons from Kanazawa University have been visiting researchers at AntiCancer, developing SOI mouse models of cancer to visualize cancer cells in vivo, tracking all stages of metastasis in real time. The present perspective review summarizes this seminal work, which has revolutionized the field of metastasis research.
Keywords: Microsurgery, mouse models, orthotopic, surgical orthotopic implantation, SOI, metastasis, fluorescent reporter gene, GFP, RFP, cancer-cell trafficking, cancer-cell-immune-cell interaction, imaging, real time, review

In 1969, one of the most important discoveries in the history of cancer research was made: athymic nude nu/nu mice could support the growth of human cancer cells (1). Until this discovery, there was no systematic experimental animal system to study human cancer. The nude mouse was subsequently demonstrated to enable growth of all types of human cancer cell lines as well as patient tumors (2). The initial nude-mouse cancer models involved subcutaneous implantation, in which metastasis rarely occurred (2). Sordat et al. (3) established the first orthotopic nude mouse model by injection of colon cancer cells into the colon of nude mice, which demonstrated cancer-cell invasion, but no metastasis.

Our laboratory began to develop orthotopic nude mouse models in the late 1980’s (4). In 1990, a breakthrough occurred when Dr. Sun Lee, the pioneer of experimental microsurgery (5), visited the laboratory at AntiCancer Inc. to demonstrate microsurgery on mice. This technique involved implanting tumor fragments, either from clinical sources or grown subcutaneously in nude mice, into the corresponding organs of other nude mice. This method became known as surgical orthotopic implantation (SOI) and led to clinical transplantation of tumors to the liver, kidney, lung, colon, breast and other organs.

Very soon, surprising results were obtained where a bladder cancer cell line was shown to be highly metastatic when transplanted by SOI in the bladder, compared to when cell suspensions were injected into the bladder (6,7). Similar results of high rates of metastasis were observed with SOI nude mouse models of the lung (8), breast (9), ovary (10), pancreas (11), colon (12), and prostate (13).

Another breakthrough occurred in the AntiCancer Laboratory when it was discovered that cancer cells expressing the jelly fish green fluorescent protein (GFP) could be imaged in vivo, even non-invasively (14,15).

At this time in 2001, one of us (NY) arrived at AntiCancer to develop imageable SOI metastatic models using GFP. The initial results and those of subsequent scientists from Kanazawa University visiting AntiCancer, are summarized below. These findings led to the development of new disruptive and revolutionary technology for the study of metastatic cancer.

Determining Clonality of Metastases in the Lungs of Mice With Fluorescent Reporter Gene

Mixtures of GFP- and red fluorescent protein (RFP)-expressing HT1080 fibrosarcoma cells were injected into the tail vein (experimental metastasis) or foot pad (spontaneous metastasis) of nude or SCID mice. Colonies that developed in the lung that were pure GFP or pure RFP, were considered clonal. Colonies of mixed GFP and RFP fluorescence were considered not clonal. Rare spontaneous lung metastasis that arose after foot-pad injection were mostly single color, thereby clonal. The frequent lung colonies arising after tail-vein injection of cell mixtures were predominantly of mixed color, not clonal. These results suggested that rare lung metastases, which are the usual clinical pattern, are clonal (Figure 1A-C) (16,17).

Imaging Metastatic Progression in the Lung With GFP- and RFP-expressing Cancer Cell Lines in Real Time

Similar to the experiments described above, mixtures of GFP and RFP cancer cells were injected into the tail vein or foot pad to develop experimental or spontaneous metastasis, respectively, but in this case to image metastatic progression in real time. Imaging was performed by visualizing the colonies growing in the lung via a skin flap over the transparent chest wall. Pure and mixed colonies growing in the lung could be seen growing in real time, indicating clonal and non-clonal metastasis, respectively (Figure 2A and B, Figure 3A-F) (17-19).

Imaging Nuclear-cytoplasmic Dynamics of Cancer Cells Expressing GFP in the Nucleus and RFP in the Cytoplasm Trafficking in Microvessels in the Brain

After common-carotid-artery injection in nude mice, cancer cells expressing GFP in the nucleus and RFP in the cytoplasm were visualized through the transparent skull with a skin flap in the scalp, in micro vessels in the brain trafficking in single-file. Both the nuclei and cytoplasm appeared deformed, and the cancers cells became elongated in order to traffic in the very narrow micro vessels of the brain. The dual-color cancer cells were so bright, that a mitotic cell could be imaged non-invasively in the ear of the mouse with the nuclei and cytoplasm of the 2 cells clearly distinguishable (Figure 4A-C) (20).

Imaging of Narrow-vessel Trafficking of Deformed Cancer Cells Labeled in the Nucleus With GFP and Cytoplasm With RFP in the Abdominal Area

With cancer cells labeled with GFP in the nucleus and RFP in the cytoplasm, it was possible, after injection of the cells into the heart, to visualize the dynamics of nuclear and cytoplasmic deformation, as well as the velocity of cancers cells trafficking in various-sized vessels in the inside surface of an abdominal skin flap. Before cancer-cell injection, the epigastrica cranialis vein in the skin flap was closed with a 6-0 suture.

The cytoplasm and nuclei of the cancer cells elongated to fit within the capillary they occupied. The cytoplasm could deform extensively, up to four times its normal length, and could fracture if the deformation was too extensive. The nuclei were more rigid and much less deformable than the cytoplasm and could extend to only 1.6 times their normal length, in narrow capillaries. In capillaries of approximately 8 μm in diameter, cancer cells could migrate 48.3 μm/h. The minimum capillary diameter that allowed cell migration was approximately 8 μm (Figure 5A-G) (21).

Imaging the Process of Extravasation of Dual-color Cancer Cells, With GFP in the Nucleus and RFP in the Cytoplasm, in Vessels Where they Are Trafficking

Cancer cells expressing GFP in the nucleus and RFP in the cytoplasm were injected through a vascular route in an abdominal skin flap and imaged in real time. The cancer cells migrated by various means in the vessels or adhered to the vessel walls and some adhered to the vessel outer surface surface after extravasation. During extravasation the cancer cells exited the vessels with the nuclei following a cytoplasmic projection that perforated the blood vessel to begin the exit process from the vessel. Both cytoplasm and nuclei underwent deformation during extravasation. Extravasation was rare for HT1080-GFP-RFP-cells but frequent for the mouse mammalian tumor cell line MMT-GFP-RFP. The trafficking cancer cells were observed to aggregate, and aggregates could collide and adhere to each other in large vessels. Cancer cells migrated at 24 μm/second in large vessels.

Post extravasation, Lewis lung cancer (LLC) -GFP-RFP cells remained closely associated with the outer vessel wall and migrated and surrounded the vessel to occupy as much of the vessel surface as possible by elongating their cytoplasm and nuclei (Figure 6A-K) (22).

Cyclophosphamide Pre-treatment Enhances Intravascular and Extravascular Growth of Cancer Cells

HT1080 fibrosarcoma cells expressing GFP and RFP died rapidly after injection into the epigastric cranialis vein in an abdominal skin flap made in nude mice. However, if 24 h before cell injection, the mice were pre-treated with the cancer-chemotherapy drug cyclophosphamide, the HT-1080-GFP-RFP cells grew extensively within blood vessels of the cyclophosphamide pre-treated mice and extravasated at high frequency and formed large extravasated colonies. It was speculated that a host-based cancer-cell killing process was inhibited by cyclophosphamide (Figure 7A-L) (23).

Imaging of Nucleolar Dynamics During the Cell Cycle

HT-1080-GFP-RFP cells were seeded on the surface of a skin flap of nude mice, and the cells were imaged 24 h after seeding. The nucleoli of the dual color cells could be imaged very clearly on the surface of the skin flap. The nucleoli were not labeled with GFP or RFP and could be clearly visualized by contrast to the GFP expression in the nucleus. When the cancer cells were in mitosis, the chromatin was highly condensed, and the nucleoli could not be visualized. After mitosis, in early G1, 4 small nucleoli were visualized in each nucleus. During the subsequent phases of the cell cycle, the nucleoli increased in size, but decreased in number. The nucleoli are a potential marker of the cell-cycle phase of cancer cells, which is important since cytotoxic chemotherapy drugs depend on the S-phase of the cell cycle for efficacy (Figure 8A-F, A’-F’) (24).

Imaging of the Interaction of Cancer Cells With Immune Cells In Vivo

Currently “immuno-oncology” is a very active area of clinical research for the treatment of cancer. Previously, we developed a relevant imageable mouse model to study the efficacy of immune-oncology agents. The model used HT1080-GFP-RFP human fibrosarcoma cells and transgenic mice expressing GFP in all immune-cell types. HT-1080-GFP-RFP cells were sprinkled on a skin flap of a GFP-transgenic mouse. After 24 h, GFP-expressing macrophages could be imaged contacting the HT1080-GFP-RFP cells and phagocytizing them. Macrophages could be imaged digesting the HT1080-GFP-RFP cells because RFP cytoplasmic fragments could be visualized within the GFP macrophages. T-cells could kill the dual-color cancer cells by fragmenting them. GFP expressing nuclei of HT1080-GFP-RFP cells which were confronted with T-cells were fragmented and appeared to be at an early stage of apoptosis. This model can be used to determine whether immuno-oncology agents, such as immune check-point inhibitors, could enhance caner-cell killing (Figure 9A-J) (25).

Imaging Real-time Shedding and Trafficking of Cancer Cells in Lymphatic Channels

Lymphatic trafficking is a common route of metastatic spread of HT1080-GFP-RFP fibrosarcoma cells. In one experiment HT1080-GFP-RFP cells were injected into nude mice in the inguinal lymph node in a skin flap, along with FITC dextran to label lymphatic channels, enabling the imaging of labeled cancer cells in lymphatic channels. The cancer cells could be imaged later in the axillary lymph nodes, also labeled with FITC dextran. Spontaneous lymphatic trafficking of cancer cells was also observed, after HT-1080-GFP-RFP cells were injected into the footpad of mice from where they subsequently metastasized to the popliteal lymph node. When the metastatic popliteal lymph node was exposed, dual-color cancer cell trafficking in lymphatic channels connected to the metastatic lymph node could be imaged. When 25 g or 100 g weights were applied to a footpad tumor formed from HT-1080-RFP-GFP cells, cancer cells were imaged shedding in the lymphatic channel leading to the popliteal lymph node, with the greater weight causing much more cancer-cell shedding than the lesser weight (Figure 10A-N) (26).

Imaging Single Cancer-cell Dynamics of Metastasis to the Lung in Real Time

In order to image the trafficking of cancer cells to the lung, the chest wall of the nude mouse had to be open for long periods. Thus, assisted breathing was necessary to keep the mice alive. Therefore, a novel retrograde wire-guided endotracheal intubation procedure for mice was developed. An intravenous catheter of 25 mm length was used as an endotracheal tube. A guide wire was inserted into the trachea through a small hole. The endotracheal catheter could then be accurately introduced into the trachea over the guide wire. The intubation tube was connected to an oxygen tube. At this point, the chest wall could be opened. Anesthesia was maintained with isoflurane applied via a vaporizer. A positive end expiratory pressure (PEEP) system was used to regulate lung inflation and deflation. After each imaging session, the chest wall was closed with 6-0 sutures. Imaging sessions were carried out for as long as 8 h and repeated up to 6 times per mouse. Cancer cells expressing GFP in the nuclei and RFP in the cytoplasm were injected into the tail vein, and HT1080-GFP-RFP or MMT-GFP-RFP cells could be imaged arriving to the lung in real time. Surviving cells in the lung could be imaged to form colonies in the lung with multiple imaging sessions over 10 days. Only rare cells among these arriving in the lung after tail vein injection could survive and form colonies in the lung (Figure 11A-Q) (27).

Imaging Bone Marrow Metastasis by Prostate Cancer Cells in Real Time

PC-3 human prostate cancer cells were injected into the heart ventricle of nude mice. Subsequently multiple metastasis were observed in the skull, femur, rib, and vertebral bones. Time-course imaging via skin flaps demonstrated the formation of metastatic colonies in the bones. Twenty min after cardiac injection of PC-3 GFP cells in RFP transgenic mice, the PC-3-GFP cells could be imaged at the single-cell level in the bone marrow of the skull.

After intra-tibial injection of PC-3 cells, RFP-expressing PC-3 cells were imaged growing in the bone marrow and spreading to an inguinal lymph node. This model can thus be used to image the lethal aspect of prostate cancer, metastasis to the bone (Figure 12A-C) (28).

Discussion

Exquisite application of experimental microsurgery and fluorescent genetic reporters enabled the establishment of unique mouse models of cancer, that along with appropriate imaging technologies, could be used to visualize all the critical aspects of cancer progression, in particular the mechanism of metastatic spread at the single-cell or sub-cellular level that distinguished the cellular cytoplasm and nuclei in vivo.

Imaging of the cancer cells was enabled by their strong expression of GFP and RFP, including the capability of cancer cells to deform and traffic in narrow vessels, extravasate, and subsequently form colonies in distant organs, all of which are the most critical steps of metastases.

Real-time in vivo imaging demonstrated the interaction of cancer cells and immune cells which can be a model of the mode of action of immune checkpoint inhibitors (ICI).

The models described in this brief perspective represent a revolutionary and disruptive improvement over previous cancer models. The unique collaboration of young physician-scientists from Kanazawa University, AntiCancer Inc., and UCSD made this possible.

Conflicts of Interest

The Authors declare no competing interests.

Authors’ Contributions

SM, NY, and RMH wrote the paper. KY, KH, HK, SM, KI, TH, HT, and SD critically read and approved the final manuscript.

Acknowledgements

This paper is dedicated to the memory of A. R. Moossa, MD, Sun Lee, MD, Professor Gordon H. Sato, Professor Li Jiaxi, Masaki Kitajima, MD, Shigeo Yagi, PhD, Jack Geller, MD, Joseph R. Bertino, MD, J.A.R. Mead PhD, Eugene P. Frenkel, MD, Professor Sheldon Penman, Professor John R. Raper and Joseph Leighton, MD.

References

1 Rygaard J & Povlsen CO Heterotransplantation of a human malignant tumour to “Nude” mice. Acta Pathol Microbiol Scand. 77(4) 758 - 760 1969. DOI: 10.1111/j.1699-0463.1969.tb04520.x
2 Hoffman RM Patient-derived mouse models of cancer. New York, NY, USA, Humana Press. DOI: 10.1007/978-3-319-57424-0
3 Sordat BCM Ueyama Y & Fogh J Metastasis of tumor xenografts in the nude mice. In: The Nude Mouse in Experimental and Clinical Research. Fogh J, Giovanella BC (eds.). Academic press.
4 Hoffman RM Patient-derived orthotopic xenografts: better mimic of metastasis than subcutaneous xenografts. Nat Rev Cancer. 15(8) 451 - 452 2015. DOI: 10.1038/nrc3972
5 Lee S Experimental Microsurgery. Tokyo, Igaku-Shoin Medical Publishers.
6 Fu X Theodorescu D Kerbel RS & Hoffman RM Extensive multi-organ metastasis following orthotopic onplantation of histologically-intact human bladder carcinoma tissue in nude mice. Int J Cancer. 49(6) 938 - 939 1991. DOI: 10.1002/ijc.2910490623
7 Fu X & Hoffman RM Human RT-4 bladder carcinoma is highly metastatic in nude mice and comparable to ras-H-transformed RT-4 when orthotopically onplanted as histologically intact tissue. Int J Cancer. 51(6) 989 - 991 1992. DOI: 10.1002/ijc.2910510625
8 Wang X Fu X & Hoffman RM A new patient-like metastatic model of human lung cancer constructed orthotopically with intact tissue via thoracotomy in immunodeficient mice. Int J Cancer. 51(6) 992 - 995 1992. DOI: 10.1002/ijc.2910510626
9 Fu X Le P & Hoffman RM A metastatic orthotopic-transplant nude-mouse model of human patient breast cancer. Anticancer Res. 13(4) 901 - 904 1993.
10 Fu X & Hoffman RM Human ovarian carcinoma metastatic models constructed in nude mice by orthotopic transplantation of histologically-intact patient specimens. Anticancer Res. 13(2) 283 - 286 1993.
11 Fu X Guadagni F & Hoffman RM A metastatic nude-mouse model of human pancreatic cancer constructed orthotopically with histologically intact patient specimens. Proc Natl Acad Sci USA. 89(12) 5645 - 5649 1992. DOI: 10.1073/pnas.89.12.5645
12 Fu XY Besterman JM Monosov A & Hoffman RM Models of human metastatic colon cancer in nude mice orthotopically constructed by using histologically intact patient specimens. Proc Natl Acad Sci USA. 88(20) 9345 - 9349 1991. DOI: 10.1073/pnas.88.20.9345
13 Fu X Herrera H & Hoffman RM Orthotopic growth and metastasis of human prostate carcinoma in nude mice after transplantation of histologically intact tissue. Int J Cancer. 52(6) 987 - 990 1992. DOI: 10.1002/ijc.2910520626
14 Chishima T Miyagi Y Wang X Yamaoka H Shimada H Moossa AR & Hoffman RM Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression. Cancer Res. 57(10) 2042 - 2047 1997.
15 Yang M Baranov E Jiang P Sun FX Li XM Li L Hasegawa S Bouvet M Al-Tuwaijri M Chishima T Shimada H Moossa AR Penman S & Hoffman RM Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases. Proc Natl Acad Sci USA. 97(3) 1206 - 1211 2000. DOI: 10.1073/pnas.97.3.1206
16 Yamamoto N Yang M Jiang P Xu M Yamauchi K Tsuchiya H Tomita K Moossa AR & Hoffman RM Color coding cancer cells with fluorescent proteins to visualize in vivo cellular interaction in metastatic colonies. Anticancer Res. 24(6) 4067 - 4072 2004.
17 Yamamoto N Yang M Jiang P Xu M Tsuchiya H Tomita K Moossa AR & Hoffman RM Determination of clonality of metastasis by cell-specific color-coded fluorescent-protein imaging. Cancer Res. 63(22) 7785 - 7790 2003.
18 Yamamoto N Yang M Jiang P Xu M Tsuchiya H Tomita K Moossa AR & Hoffman RM Real-time imaging of individual fluorescent-protein color-coded metastatic colonies in vivo. Clin Exp Metastasis. 20(7) 633 - 638 2003. DOI: 10.1023/a:1027311230474
19 Yamamoto N Yang M Jiang P Tsuchiya H Tomita K Moossa AR & Hoffman RM Real-time GFP imaging of spontaneous HT-1080 fibrosarcoma lung metastases. Clin Exp Metastasis. 20(2) 181 - 185 2003. DOI: 10.1023/a:1022662927574
20 Yamamoto N Jiang P Yang M Xu M Yamauchi K Tsuchiya H Tomita K Wahl GM Moossa AR & Hoffman RM Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-color nuclear-cytoplasmic fluorescent-protein expression. Cancer Res. 64(12) 4251 - 4256 2004. DOI: 10.1158/0008-5472.CAN-04-0643
21 Yamauchi K Yang M Jiang P Yamamoto N Xu M Amoh Y Tsuji K Bouvet M Tsuchiya H Tomita K Moossa AR & Hoffman RM Real-time in vivo dual-color imaging of intracapillary cancer cell and nucleus deformation and migration. Cancer Res. 65(10) 4246 - 4252 2005. DOI: 10.1158/0008-5472.CAN-05-0069
22 Yamauchi K Yang M Jiang P Xu M Yamamoto N Tsuchiya H Tomita K Moossa AR Bouvet M & Hoffman RM Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable-magnification whole-mouse imaging system. Cancer Res. 66(8) 4208 - 4214 2006. DOI: 10.1158/0008-5472.CAN-05-3927
23 Yamauchi K Yang M Hayashi K Jiang P Yamamoto N Tsuchiya H Tomita K Moossa AR Bouvet M & Hoffman RM Induction of cancer metastasis by cyclophosphamide pretreatment of host mice: an opposite effect of chemotherapy. Cancer Res. 68(2) 516 - 520 2008. DOI: 10.1158/0008-5472.CAN-07-3063
24 Yamauchi K Yang M Hayashi K Jiang P Yamamoto N Tsuchiya H Tomita K Moossa AR Bouvet M & Hoffman RM Imaging of nucleolar dynamics during the cell cycle of cancer cells in live mice. Cell Cycle. 6(21) 2706 - 2708 2007. DOI: 10.4161/cc.6.21.4861
25 Yamauchi K Tome Y Yamamoto N Hayashi K Kimura H Tsuchiya H Tomita K Bouvet M & Hoffman RM Color-coded real-time subcellular fluorescence imaging of the interaction between cancer and host cells in live mice. Anticancer Res. 32(1) 39 - 43 2012.
26 Hayashi K Jiang P Yamauchi K Yamamoto N Tsuchiya H Tomita K Moossa AR Bouvet M & Hoffman RM Real-time imaging of tumor-cell shedding and trafficking in lymphatic channels. Cancer Res. 67(17) 8223 - 8228 2007. DOI: 10.1158/0008-5472.CAN-07-1237
27 Kimura H Hayashi K Yamauchi K Yamamoto N Tsuchiya H Tomita K Kishimoto H Bouvet M & Hoffman RM Real-time imaging of single cancer-cell dynamics of lung metastasis. J Cell Biochem. 109(1) 58 - 64 2010. DOI: 10.1002/jcb.22379
28 Miwa S Toneri M Igarashi K Yano S Kimura H Hayashi K Yamamoto N Tsuchiya H & Hoffman RM Real-time in vivo confocal fluorescence imaging of prostate cancer bone-marrow micrometastasis development at the cellular level in nude mice. J Cell Biochem. 117(11) 2533 - 2537 2016. DOI: 10.1002/jcb.25545
cdp > Vol 4 - 5 > Pages 544 - 557