Open Access

Imaging Transgenic Nude Mice Expressing Spectrally-distinct Fluorescent Reporters Emitting From Blue to Far Red Light With One Instrument With Single-nanometer-tuning of Laser-excitation Fluorescence

MIZUTA KOHEI 1 2
REYNOSO JOSE 1
GALLAGHER SEAN 3
WANG APRIL 3
CHANG NEIL 3
MORINAGA SEI 1 2
SATO MOTOKAZU 1 2
KANG BYUNG MO 1 2
  &  
HOFFMAN ROBERT M. 1 2

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

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

3Analytik Jena, Upland, CA, U.S.A.

Cancer Diagnosis & Prognosis Sep-Oct; 4(5): 563-566 DOI: 10.21873/cdp.10364
Received 13 May 2024 | Revised 03 October 2024 | Accepted 26 June 2024
Corresponding author
Robert M. Hoffman, Ph.D., AntiCancer, Inc., 7917 Ostrow Street, San Diego, CA 92111, U.S.A. Tel: +1 6198852284, email: all@anticancer.com

Abstract

Background/Aim: Transgenic nude mice expressing green fluorescent protein (GFP), red fluorescent protein (RFP), or cyan fluorescent protein (CFP) were previously developed by our laboratory, AntiCancer Inc. In the present study, we demonstrate imaging of the GFP, RFP, or CFP nude mice with single-nanometer-tuning laser fluorescence excitation with a single instrument. Materials and Methods: Female transgenic C57/B6 nude GFP, RFP, and CFP mice aged six weeks were used. The images were obtained using the UVP Biospectrum Advanced system (Analytik Jena US LLC) with excitation at 480 nm and peak emission at 513 nm for GFP; 520 nm and 605 nm, respectively, for RFP; and 405 nm and 480 nm, respectively, for CFP. Results: For each color transgenic fluorescent mouse, images without background could be obtained individually with the UVP Biospectrum Advanced system. Conclusion: Using a single instrument, brilliant and well-defined images of GFP, RFP, and CFP mice were obtained with single-nanometer-tuning laser fluorescence excitation. This imaging system will be used in future studies to analyze cancer cells in the colored mice that are spectrally distinct in order to determine how stromal cells and cancer interact in the tumor microenvironment.
Keywords: Laser excitation, single nanometer tuning, fluorescence imaging, transgenic nude mice, GFP, RFP, CFP

Transgenic green fluorescent protein (GFP), red fluorescent protein (RFP), or cyan fluorescent protein (CFP) nude mice were previously developed at our laboratory, AntiCancer Inc. (1-3). Fluorescent-protein-imaging in vivo, pioneered by our laboratory, is used to investigate tumor growth and progression at the macro, cellular, and subcellular levels in vivo (4-8). Using spectrally-distant fluorescent proteins, cancer and stromal cells could be distinguished by multi-color imaging in the tumor microenvironment (TME) (1,3,4,9-13).

In the present study, we demonstrate imaging of transgenic nude mice with spectrally-distinct fluorescent reporters expressing GFP, RFP, or CFP, with single-nanometer-tuning laser fluorescence excitation using one instrument.

Materials and Methods

Mice. Transgenic C57/B6 nude mice expressing GFP, RFP, or CFP aged six weeks (AntiCancer, Inc., San Diego, CA, USA) were used (Figure 1). The cytomegalovirus enhancer and the chicken β-actin promoter regulate the expression of the fluorescent protein genes in these transgenic nude mice (1-3). All mice were bred at AntiCancer Inc. in a barrier facility with a high efficiency particulate air (HEPA)-filtered rack, and kept in standard settings with 12-h light/dark cycles. The present protocol was approved by the AntiCancer Inc. Institutional Animal Care and Use Committee following the National Institutes of Health Guide for the Care and Use of Animals.

Imaging. The present study used a UVP Biospectrum Advanced system (Analytik Jena US LLC) (Figure 2) for imaging with single-nanometer-wavelength tuning of laser-excitation fluorescence, at 480 nm and peak emission at 513 nm for GFP; 520 nm and 605 nm, respectively, for RFP; and 405 nm and 480 nm, respectively, for CFP. Imaging was performed using an anesthetic solution of 50% ketamine (100 mg/ml), 38% xylazine (100 mg/ml), and 12% acepromazine maleate (10 mg/ml) injected intramuscularly.

Results

Bright-field images of a transgenic CFP nude mouse; an RFP transgenic nude mouse and a GFP transgenic nude mouse are shown in Figure 1. The expression of RFP is so strong in the transgenic RFP nude mouse that it appears red even in bright light. Merged very bright images of transgenic GFP, RFP, and CFP nude mice were obtained with single-nanometer-tuning fluorescence laser excitation (Figure 3).

Discussion

The present study described a single instrument (Analytik Jena UVP Biospectrum Advanced system) with single-nanometer-tuning laser fluorescence excitation, that could image brightly and clearly transgenic mice expressing fluorescent reporters emitting over a broad range of wavelengths from 480 nm to 605 nm with excitement ranging from 405 nm to 520 nm.

The application of fluorescent proteins to in vivo imaging is routinely used to monitor the growth and progression of tumors at the macro, cellular, and subcellular levels (4-8).

In vivo imaging with fluorescent proteins was pioneered by our laboratory (7-9).

Imaging the interaction of cancer and stromal cells in the TME using spectrally-distinct fluorescent proteins has been demonstrated (1,3,4,9-13). Bouvet et al. observed the tumor-host interaction in liver metastasis using transgenic nude mice expressing GFP and human colon-cancer cell lines expressing RFP (12). Suetsugu et al. demonstrated pancreatic-cancer-patient tumors established in NOD/SCID mice that were subsequently passaged orthotopically in transgenic nude mice expressing RFP, acquired stroma expressing RFP. Further passage to transgenic nude mice expressing GFP and CFP resulted in the tumors acquiring GFP and CFP stroma, respectively. Multicolored stroma are useful in analyzing the role of host stroma in the initiation and progression of metastasis (13).

Recent studies have used single-nanometer-tuning laser excitation to image orthotopic tumors expressing fluorescent reporters without background (14-16).

In the present study, multi-spectral in vivo imaging with single-nanometer-tuning (14-16) was performed on nude mice expressing transgenic fluorescent protein with emission ranging from 480 to 605 nm.

Conclusion

The very narrow-bandwidth-tuning laser excitation enabled the acquisition of very bright and well-defined images of GFP, RFP, and CFP transgenic mice with a single instrument. Further studies will use this imaging system to study spectrally-distinct cancer cells in each colored mice to determine the interaction of cancer and stromal cells in the TME.

Conflicts of Interest

AW, NC, and SG are employees of Analytik Jena. JR is an employee of Anticancer Inc. KM, SM, MS, BMK, and RMH are non-salaried associates of AntiCancer Inc. AntiCancer Inc. uses mouse models of cancer for contract research.

Authors’ Contributions

KM, SG, AW, and NC performed experiments. KM and RMH wrote this article. JR provided the transgenic nude mice, SM, MS, and BMK, critically reviewed this article.

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, and Professor John R. Raper and Joseph Leighton MD. The Robert M. Hoffman Foundation for Cancer Research provided funds for this study.

Funding

The Robert M. Hoffman Foundation for Cancer Research contributed to the funding of this study.

References

1 Yang M Reynoso J Bouvet M & Hoffman RM A transgenic red fluorescent protein-expressing nude mouse for color-coded imaging of the tumor microenvironment. J Cell Biochem. 106(2) 279 - 284 2009. DOI: 10.1002/jcb.21999
2 Tran Cao HS Reynoso J Yang M Kimura H Kaushal S Snyder CS Hoffman RM & Bouvet M Development of the transgenic cyan fluorescent protein (CFP)-expressing nude mouse for “Technicolor” cancer imaging. J Cell Biochem. 107(2) 328 - 334 2009. DOI: 10.1002/jcb.22128
3 Yang M Reynoso J Jiang P Li L Moossa AR & Hoffman RM Transgenic nude mouse with ubiquitous green fluorescent protein expression as a host for human tumors. Cancer Res. 64(23) 8651 - 8656 2004. DOI: 10.1158/0008-5472.CAN-04-3118
4 Hoffman RM & Yang M Color-coded fluorescence imaging of tumor-host interactions. Nat Protoc. 1(2) 928 - 935 2006. DOI: 10.1038/nprot.2006.119
5 Hoffman RM & Yang M Subcellular imaging in the live mouse. Nat Protoc. 1(2) 775 - 782 2006. DOI: 10.1038/nprot.2006.109
6 Hoffman RM The multiple uses of fluorescent proteins to visualize cancer in vivo. Nat Rev Cancer. 5(10) 796 - 806 2005. DOI: 10.1038/nrc1717
7 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.
8 Hoffman RM & Yang M Whole-body imaging with fluorescent proteins. Nat Protoc. 1(3) 1429 - 1438 2006. DOI: 10.1038/nprot.2006.223
9 Yang M Li L Jiang P Moossa AR Penman S & Hoffman RM Dual-color fluorescence imaging distinguishes tumor cells from induced host angiogenic vessels and stromal cells. Proc Natl Acad Sci USA. 100(24) 14259 - 14262 2003. DOI: 10.1073/pnas.2436101100
10 Suetsugu A Osawa Y Nagaki M Saji S Moriwaki H Bouvet M & Hoffman RM Imaging the recruitment of cancer-associated fibroblasts by liver-metastatic colon cancer. J Cell Biochem. 112(3) 949 - 953 2011. DOI: 10.1002/jcb.23011
11 Suetsugu A Hassanein MK Reynoso J Osawa Y Nagaki M Moriwaki H Saji S Bouvet M & Hoffman RM The cyan fluorescent protein nude mouse as a host for multicolor-coded imaging models of primary and metastatic tumor microenvironments. Anticancer Res. 32(1) 31 - 38 2012.
12 Bouvet M Tsuji K Yang M Jiang P Moossa AR & Hoffman RM In vivo color-coded imaging of the interaction of colon cancer cells and splenocytes in the formation of liver metastases. Cancer Res. 66(23) 11293 - 11297 2006. DOI: 10.1158/0008-5472.CAN-06-2662
13 Suetsugu A Katz M Fleming J Truty M Thomas R Moriwaki H Bouvet M Saji S & Hoffman RM Multi-color palette of fluorescent proteins for imaging the tumor microenvironment of orthotopic tumorgraft mouse models of clinical pancreatic cancer specimens. J Cell Biochem. 113(7) 2290 - 2295 2012. DOI: 10.1002/jcb.24099
14 Kubota Y Wang A Chang N Tarantino S Gallagher S Aoki Y Masaki N Obara K Morinaga S Tsunoda T & Hoffman RM Precise non-invasive imaging mouse model of pancreatic cancer: very narrow band-width laser fluorescence excitation of green fluorescent protein provides ultra-bright tumor images with no skin autofluorescence. Cancer Diagn Progn. 4(1) 30 - 33 2024. DOI: 10.21873/cdp.10281
15 Kubota Y Aoki Y Wang A Chang N Tarantino S Gallagher S Tsunoda T & Hoffman RM Non-invasive fluorescence imaging of breast cancer metastasis to the brain in an orthotopic nude-mouse model with very-narrow-band-width laser excitation of red fluorescent protein resulting in an ultra-bright signal without skin autofluorescence. In Vivo. 38(1) 69 - 72 2024. DOI: 10.21873/invivo.13411
16 Mizuta K Gallagher S Wang A Chang N Morinaga S Sato M Kang BM & Hoffman RM Head-to-head comparison of green fluorescent protein (GFP) imaging with luciferase-luciferin imaging in vivo using single-nanometer laser-excitation tuning and an ultra-low-light-detection camera and optics demonstrates the superiority of GFP. Anticancer Res. 44(7) 2823 - 2826 2024. DOI: 10.21873/anticanres.17094