Introduction

Non-invasive Detection of a SingleBioluminescent 4T1 Cell In Vivo

Detection of tumors and quantitation of tumor burdens by bioluminescence and fluorescence imaging has made significant progress over the past several years. Due to tissue absorption properties and low tissue autoluminescence, bioluminescence imaging has been adopted as a main non-invasive technology to monitor tumor cells in preclinical settings (1). Stable and bright bioluminescent cell lines made possible detection of few cancer cells in live animals. This facilitates monitoring of growth and responses to treatments of small metastasis, and also allows tracking of tumor behavior right from the time of implantation before the tumors are palpable or can be detected by any other imaging modality. In addition, brighter cells are desired in orthotopic tumor models, where injection volumes are often limited and the depth of tissues covering the tumors can significantly attenuate emitted light. Implanting brighter tumor cells improves the chances of detecting micrometastases sooner.

Most of the previous generations of luciferase labeled cell lines have luciferase expression levels resulting in light emission of less than 100 photons/sec/cell. Recently, we have developed a series of super bright tumor cell lines using enhanced luciferase 2 (luc2) gene stably expressed from the human Ub C promoter (2). As an example, the 4T1 mouse mammary tumor cell line was transfected using luc2 vector and stable clones were isolated using puromycin selection (3). Our first round of cloning resulted in 8 bright clones that initially emitted more than 50,000 photons/sec/cell, and which light emission decreased to around 3,000 photons/sec/cell in the second week and remained stable at that level in vitro for the following 4 weeks without any selection pressure. After implanting the cells from the above clones into SCID-bg mice we showed that we could detect as little as 100 cells in vivo. However, prolonged in vitro culturing of the clones resulted in the decline of luciferase activity. Therefore, we attempted a second round of single cell cloning process and generated additional 4 clones. Like in the first round of cloning, the resulting clones showed more than 50,000 photons/sec/cell light emission in the first week of the stability testing. Subsequently, their luciferase activities decreased in week 2, but then stabilized giving the light emission of about 7,000 ~ 10,000 photons/sec/cell. One clone was chosen for further analyses (4T1-luc2-1A4).

Since a cell uses 1 ATP molecule to produce a photon of light, extremely high levels of light emission could potentially be detrimental to the cell’s metabolism due to the possible depletion of the cell’s ATP pool.

To test whether high light production affects cell physiology, we observed cell growth for 4 days in the presence of high levels of D-luciferin (300 μg/mL/day, added daily in two equal doses). Our results indicate that high light emission does not affect cell growth, and that clones expressing high levels of luciferase showed the same growth characteristics in the presence and absence of D-luciferin. Moreover the growth of these cells was comparable to that of the parental 4T1 cells. To determine the detection limit of bioluminescent cells in vivo, we subcutaneously implanted series of small numbers of 4T1-luc2-1A4 cells (3, 5, 10 and 50 cells) into nu/nu mice. The total flux (light emission) from implantation sites was directly proportional to the numbers of cells implanted. We were able to detect as little as 3 cells in the animal using a highly sensitive CCD camera. Despite harsh environment for the implanted cells in the body, many cells survived for up to 6 hours (the end of the observation time in this particular experiment). Next, we attempted to detect a single bioluminescent cell in vivo. To ensure reliable delivery of a single cell, we used a micromanipulator to deliver the single cell under the skin of the athymic, nude mouse. Bioluminescence images were taken immediately after the cell implantation. Our results clearly demonstrate that a single bioluminescent tumor cell could be detected. Interestingly, animals injected with 5 and 10 cells developed tumors over time.

Our preliminary results show that when 4T1-luc2-1A4 cells were implanted orthotopically in syngeneic mice, they develop detectable metastases in various organs. These bright cells can be used to monitor early efficacy of drug candidates in orthotopic models of breast cancer, as well as in models of metastasis. In addition the ultra bright cells can be potentially used to study development of tumors from single cancer stem cell.

Results

Results (cont)

Materials and MethodsGeneration of lentivirus vector Enhanced luciferase 2 (luc2) cDNA was from pGL4.20 vector (Promega, WI). Luciferase 2 cDNA was excised with Hind III & Xba I and ligated into pUB6-V5-HisB vector (Invitrogen, CA) (pUB6-luc2). A fragment gener-ated by Bgl II and BstB I digestion of pUB6-luc2 was ligated into a modified pLPCX vector (Clontech, CA). A lentiviral vector that carries human ubiquitin C promoter and luc2 cDNA was generated by inserting Bgl II & EcoR I fragment from above construct into Bcl I & EcoR I of the modified pLKO.1m vector (Sigma-Aldrich, MO) (pLPU-luc2) (4).

Cell Culture and growth curve generationMouse mammary gland tumor cell line 4T1 was obtained from the ATCC (Manassas, VA). Cells were grown in high glucose RPMI 1640 medium (ATCC) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) without antibiotics. Growth curves were generated by seeding 100,000 cells in a T25 flask. At each time point, cells were trypsinized and counted using an automatic cell counter (Nexcelom, Lawrence, MA). Total numbers of cells were plotted in a logarithmic scale.

Transfection Lentiviral vector pLPU-luc2 was transfected using a lipid based method. Transfected cells were selected using puromycin (2 μg/mL). Isolated clones were screened for their luciferase activities using an IVIS Spec-trum (Caliper Life Sciences, Hopkinton, MA). To isolate single cell clones, cells were subjected to limited dilu-tion. Individual clones were screened for luciferase activity using an IVIS Spectrum. Selected clones were maintained without puromycin for 4 weeks.

Animals and tumor cell implantationAll the procedures for animal care and tumor cell implantation followed the approved animal protocols and guidelines of the Institutional Animal Care and Use Committee. Prior to implantation, all tumor cells were tested for the presence of mycoplasma and mouse pathogens. Female nu/nu mice (Charles River, Wilming-ton, MA) were anesthetized with isoflurane and were injected with 4T1-luc2-1A4 cells subcutaneously below the dorsal flank. To ensure one cell injection, a micromanipulator was used. Cells were plated on a plate im-mediately after trypsinization. Cells were picked up less than 10 μL of volume using a micromanipulator. Two slits were made on the mouse back prior to injection. Cells were inserted into the slits and pipets were exam-ined under the microscope to make it sure there was no cells left in pipets. Bioluminescence images were taken 10 min after luciferin injection.

In vitro and in vivo bioluminescence imaging For in vitro luciferase assay, cells were plated on black walled 24-well plates at an initial density of 50,000 cells/well. Cells were grown overnight with regular growth medium. After 24 hr, the medium was replaced with D-luciferin containing medium (150 μg/mL). Bioluminescence images were taken immediately after adding the substrate into the cells using an IVIS Spectrum. Light outputs were quantified using Living Image software version 3.0 (Caliper Life Sciences, Alameda, CA). For in vivo imaging, the mice were anesthetized with isoflu-rane. D-luciferin solution was injected intraperitoneally (150 mg/kg). Mice were subjected to in vivo biolumi-nescence imaging using an IVIS Spectrum, and the results were quantified using Living Image software.

Acknowledgements - The authors thank Drs. Ning Zhang, Victor Ninov, Chaincy Kuo, and Brad Rice for their helpful discussions.

Figure 1. Generation of 4T1-luc2 cells - Mouse mammary tumor 4T1 cells were transfected with a lentiviral vector pLPU-luc2. Puromycin resistant clones were isolated and their luciferase expressions were screened by bioluminescence. Single cell cloning was performed to generate clonal lines. Cells were plated at density of 50,000 cell/well in a black-walled 24-well plate. After incubating overnight at 37 °C, regular growth media was replaced with D-luciferin containing media. Images were taken immediately after changing the media using an IVIS® Spectrum™ (Binning: med, f stop: 1, exposure time: 1 sec). Four high expressers are shown.

Figure 2. Stability of luciferase activity in 4T1-luc2 clones - To check the stability of luciferase activity, transfected 4T1-luc2 clones were monitored for their luciferase activities for 4 weeks. Cells were grown with absence of selective pressure. Bioluminescent images were taken using an IVIS Spectrum each week and Total flux (photons/sec) was quantitated using Living Image® software 3.0. Light emission per cell (photons/sec/cell) was determined. All clones emitted light of more than 7,000 photons/sec/cell.

Figure 3. Growth curves for 4T1-luc2 cells in the presence or absence of D-luciferin - (A) Growth of parental 4T1 and 4T1-luc2-1A4 cells in regular growth medium. Hundred thousands cells were plated and grown without D-luciferin. Total numbers of cells were counted at each time point and plotted in a logarithmic scale. 4T1-luc2-1A4 clone showed similar growth pattern and doubling time when compared to parental 4T1 cells. (B) Growth of parental 4T1 and 4T1-luc2-1A4 in the presence of D-luciferin (150 μg/mL/day). Cells were harvested at each time point and counted. The growth pattern of the 4T1-luc2-1A4 in the presence of D-luciferin was similar to that of parental cell line. (C) Growth of parental 4T1 and 4T1-luc2-1A4 in the presence of D-luciferin (300 μg/mL/day). Presence of the excess of D-luciferin did not affect the overall growth patterns of the 4T1-luc2 cells.

Figure 4. Detection of small numbers of 4T1-luc2-1A4 cells in vivo - (A) Bioluminescent images of 4T1-luc2-1A4 implanted into flank regions of nu/nu mice. Cells were prepared by serial dilution. Numbers of cells injected into animals are listed. Images were taken im-mediately after implantation using an IVIS Spectrum. (B) Bioluminescent images were taken 6 hrs post-implantation using an IVIS Spectrum. Numbers of implanted cells are shown in each panel. Imaging conditions (FOV: C, binning: medium, f stop: 1, exposure time: 5 min).

Figure 6. Tumor growth from five and ten 4T1-luc2-1A4 cells in nu/nu mice - (A) Tumor formation from five 4T1-luc-1A4 cell implantation. Cells were subcutaneously injected into the flank region of the mouse. Bioluminescent images were taken over the time using an IVIS Spectrum. Imaging conditions are listed. (B) Monitoring of tumor growth by bioluminescence and caliper measurements. Tumor was not palpable till post-implantation day 27 while bioluminescence signal increased from implantation day 0. (C) Tumor formation from 10 cell implantation of 4T1-luc2-1A4 cells. Imaging conditions listed. (D) Monitoring of tumor growth by bioluminescence and caliper measurements.

Figure 5. Detection of a single 4T1-luc2-1A4 cell in vivo - (A-D) A single 4T1-luc2-1A4 cell was implanted into the back of a nu/nu mouse using a micromanipulator. D-luciferin was injected and bioluminescent images were taken 10 min after luciferin injection suing an IVIS Spectrum. (A) Whole mouse bioluminescent image with a single cell implantation. (B) Magnified image of dotted area in panel A. Asterisk (*); background signal from the gut, dotted circle; single cell signal. (C, D) Line profiling analysis of single cell signal. Light emission was plotted along with the line in panel C. Peak signal in panel D represents the light emission from a single 4T1-luc2-1A4 cell. (E,F) Signal from 10 4T1-luc2-1A4 cells in a nu/nu mouse. Cells were picked up and injected with a micromanipulator into the back of the mouse subcutaneously. (E) Whole mouse image with 10 cell implantation. (F) Magnified image of dotted area in panel E. Dotted circle indicates the signals from 10 cells.

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Jae-Beom Kim, Konnie Urban, Steve Lee, Adam Bata, Kenneth Campbell*,Richard Coffee*, Alex Gorodinsky*, Brad Rice, and Peter Lassota

Caliper Life Sciences, 2061 Challenger Dr, Alameda, CA 94501, *5 Cedar Brook Dr, Cranbury, NJ 08512 USA

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