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.

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.

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).

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.

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.

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.

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.

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.

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