**3.1 Engineering Luciferse expressing cells**

Luciferase positive tumor cells used for generating animal models in cancer research are widely available in almost all histological types such as breast, cervical, colorectal, lung, prostate, ovarian cancer and melanoma. Developing a luciferase expressing mammary cell line can be accomplished in house by standard transfection or transduction methodology using reagents, such as plasmid vectors carrying the luciferase genes, which are commercially available.

Tang et al use a typical transfection procedure to create a luciferase positive neuroprogenitor cell line where the pGL3Basic plasmid (Promega, Madison, WI) carrying F-Luc was digested with HindIII and BamHI and the 1.9-kb cDNA fragment encoding F-Luc was isolated and cloned into a second vector (the pHGCX). The resulting pHGCX vector contained the F-Luc gene driven by the cytomegalovirus (CMV) promoter which enables constitutive expression of luciferase. The pHGCX vector also contained the gene for enhanced green fluorescent protein (eGFP) under the control of the viral immediate early

Bioluminescence Applications in Preclinical Oncology Research 143

al reported the development of a luciferase transgenic mouse model where F-Luc was placed under the control of the Prostate Specific Antigen (PSA) promoter to develop transgenic PSA-Luc mice. Figure 2A shows the BLI of representative PSA-Luc transgenic male and female animals, and non-transgenic littermate male mice. Figure 2B shows BLI of excised organs from a 12-week-old PSA-Luc male mouse. Hsieh et al demonstrate that the PSA-Luc mouse model has luciferase based BL restricted to the prostate gland (Hsieh, Xie et al. 2005). This mouse model has been used to monitor the prostate gland during

A

B Fig. 1. Imaging luciferase positive breast cancer cells in vitro. LCC6-Luc cells were serially diluted and placed into wells of a 24 well plate. Luciferin was added and cells were

(A-representative images). These data were used to generate a plot comparing total light emission to cell number (B - graph). (Kalra, J., M. Anantha, et al. (2011). "Validating the use of a luciferase labeled breast cancer cell line, MDA435LCC6, as a means to monitor tumor progression and to assess the therapeutic activity of an established anticancer drug, docetaxel (Dt) alone or in combination with the ILK inhibitor, QLT0267." Cancer Biol Ther

immediately imaged using the IVIS 200 system to obtain BL measurements

11(9): 826-838. Reproduced by permission of author.)

development, tumorogenesis and in response to androgens or chemotherapy.

IE4/5 promoter, providing a method of fluorescence selection. The neuroprogeneitor celline (C17.2) were stably cotransfected with the engineered vector and pBabePuro, containing the gene for puromycin resistance, using Lipofectamine transfection reagent (Invitrogen Life Technologies, Carlsbad, CA) (Tang, Shah et al. 2003).

Stable luciferase expressing cells can also be generated through transduction strategies (Nyati, Symon et al. 2002; Kalra, Warburton et al. 2009; Kalra, Anantha et al. 2011; Yan, Xiao et al. 2011). Our own lab uses transduction procedures to co-express F-Luc and GFP in breast cancer cell lines. Briefly, the luciferase coding sequence was isolated from the pGL3Basic vector (Promega, Madison, WI) and cloned into the lentiviral vector, FG9, downstream of the CMV and UBiC promoters again to support constitutive activation of the F-Luc gene. The engineered vector was cotransfected with packaging constructs pRSVREV, pMDLg/pRRE and the VSV-G expression plasmid pHCMVG into a packaging cell line (HEK-293T) by a standard LipofectAMINE 2000 (Invitrogen, Burlington, ON Canada) transfection procedure. Conditioned medium containing Lentivirus-Luciferase (Lenti-Luc) particles was collected and cleared of debris by low speed centrifugation. A similar method was used to generate GFP-expressing lentivirus (Lenti-GFP). The Breast cancer cell line (MDA MB 435/LCC6 (LCC6)) was then infected with Lenti-Luc and Lenti-GFP creating LCC6-Luc/GFP cells. To enrich for luciferase positive cells, cells were sorted by FACS for GFP expression. Subsequently GFP-positive cells were re-plated in low concentrations into soft agar in the wells of a 96-well plate. Luciferin was added to each well and plates were imaged using IVIS to identify luciferase positive colonies (Kalra, Warburton et al. 2009; Kalra, Anantha et al. 2011).

#### **3.2 Use of Luciferase positive cell lines**

Once stable luciferase positive cell lines are generated, the cells should be assessed for growth rates and sensitivity to selected drugs; comparing engineered cells to parental cell lines. These assays are performed to ensure that the luciferase gene does not interfere with cell function. Many groups will also image a serial dilution of cells to associate BL produced with number of cells as shown in Figure 1. Figure 1A exhibits the CCD camera capture of a serial dilution of luciferase positive human breast cancer (LCC6-Luc) after luciferin substrate is added. BL data was quantified as photons emitted per second. The resulting graph (Figure 1B) shows that the photons emitted are proportional to the number of cells plated (Kalra, Anantha et al. 2011). These data can be used to estimate cell numbers from BL captured in an in vivo model. In this example the minimum number of cells detectable was approximately 10,000 cells, but the detection limit is dependent on multiple factors including lucifern concentration, imaging programs, parameters such as exposure time and the device used for image capture.

Once luciferase positive cells are adequately vetted in vitro, these cells can be used to establish an in vivo model of disease through multiple routes of inoculation as discussed in Section 6.

#### **3.3 Luciferase expressing transgeneic mice**

A wide variety of transgenic reporter animals using luciferase-based technology have been engineered in order to visualize transgene expression in vivo. For example, in 2005 Hsieh et

IE4/5 promoter, providing a method of fluorescence selection. The neuroprogeneitor celline (C17.2) were stably cotransfected with the engineered vector and pBabePuro, containing the gene for puromycin resistance, using Lipofectamine transfection reagent (Invitrogen Life

Stable luciferase expressing cells can also be generated through transduction strategies (Nyati, Symon et al. 2002; Kalra, Warburton et al. 2009; Kalra, Anantha et al. 2011; Yan, Xiao et al. 2011). Our own lab uses transduction procedures to co-express F-Luc and GFP in breast cancer cell lines. Briefly, the luciferase coding sequence was isolated from the pGL3Basic vector (Promega, Madison, WI) and cloned into the lentiviral vector, FG9, downstream of the CMV and UBiC promoters again to support constitutive activation of the F-Luc gene. The engineered vector was cotransfected with packaging constructs pRSVREV, pMDLg/pRRE and the VSV-G expression plasmid pHCMVG into a packaging cell line (HEK-293T) by a standard LipofectAMINE 2000 (Invitrogen, Burlington, ON Canada) transfection procedure. Conditioned medium containing Lentivirus-Luciferase (Lenti-Luc) particles was collected and cleared of debris by low speed centrifugation. A similar method was used to generate GFP-expressing lentivirus (Lenti-GFP). The Breast cancer cell line (MDA MB 435/LCC6 (LCC6)) was then infected with Lenti-Luc and Lenti-GFP creating LCC6-Luc/GFP cells. To enrich for luciferase positive cells, cells were sorted by FACS for GFP expression. Subsequently GFP-positive cells were re-plated in low concentrations into soft agar in the wells of a 96-well plate. Luciferin was added to each well and plates were imaged using IVIS to identify luciferase positive colonies (Kalra, Warburton et al. 2009;

Once stable luciferase positive cell lines are generated, the cells should be assessed for growth rates and sensitivity to selected drugs; comparing engineered cells to parental cell lines. These assays are performed to ensure that the luciferase gene does not interfere with cell function. Many groups will also image a serial dilution of cells to associate BL produced with number of cells as shown in Figure 1. Figure 1A exhibits the CCD camera capture of a serial dilution of luciferase positive human breast cancer (LCC6-Luc) after luciferin substrate is added. BL data was quantified as photons emitted per second. The resulting graph (Figure 1B) shows that the photons emitted are proportional to the number of cells plated (Kalra, Anantha et al. 2011). These data can be used to estimate cell numbers from BL captured in an in vivo model. In this example the minimum number of cells detectable was approximately 10,000 cells, but the detection limit is dependent on multiple factors including lucifern concentration, imaging programs, parameters such as exposure time and

Once luciferase positive cells are adequately vetted in vitro, these cells can be used to establish an in vivo model of disease through multiple routes of inoculation as discussed in

A wide variety of transgenic reporter animals using luciferase-based technology have been engineered in order to visualize transgene expression in vivo. For example, in 2005 Hsieh et

Technologies, Carlsbad, CA) (Tang, Shah et al. 2003).

Kalra, Anantha et al. 2011).

**3.2 Use of Luciferase positive cell lines** 

the device used for image capture.

**3.3 Luciferase expressing transgeneic mice** 

Section 6.

al reported the development of a luciferase transgenic mouse model where F-Luc was placed under the control of the Prostate Specific Antigen (PSA) promoter to develop transgenic PSA-Luc mice. Figure 2A shows the BLI of representative PSA-Luc transgenic male and female animals, and non-transgenic littermate male mice. Figure 2B shows BLI of excised organs from a 12-week-old PSA-Luc male mouse. Hsieh et al demonstrate that the PSA-Luc mouse model has luciferase based BL restricted to the prostate gland (Hsieh, Xie et al. 2005). This mouse model has been used to monitor the prostate gland during development, tumorogenesis and in response to androgens or chemotherapy.

B

Fig. 1. Imaging luciferase positive breast cancer cells in vitro. LCC6-Luc cells were serially diluted and placed into wells of a 24 well plate. Luciferin was added and cells were immediately imaged using the IVIS 200 system to obtain BL measurements (A-representative images). These data were used to generate a plot comparing total light emission to cell number (B - graph). (Kalra, J., M. Anantha, et al. (2011). "Validating the use of a luciferase labeled breast cancer cell line, MDA435LCC6, as a means to monitor tumor progression and to assess the therapeutic activity of an established anticancer drug, docetaxel (Dt) alone or in combination with the ILK inhibitor, QLT0267." Cancer Biol Ther 11(9): 826-838. Reproduced by permission of author.)

Bioluminescence Applications in Preclinical Oncology Research 145

They were able to use BL to image the growth and development of spontaneous tumors and showed that in response to treatment using doxorubicin and prednisone, these tumors were

(ER-DEVD-Luc) Apoptosis Laxman 2002 (Laxman, Hall

E2F1-Luc Cell proliferation Momota 2005(Momota and

Estrogen Receptor (ER-Luc) Hormone receptor activity Wu 2008(Wu, Xu et al. 2008)

Factor (VEGF-Luc) Angiogenesis Faley 2007(Faley, Takahashi

Survivin (Survivin –Luc) Apoptosis Li 2010(Li, Cheng et al. 2010)

Collagen1 alpha 1 (Col-Luc) Bone metastasis Lee 2010(Lee, Huang et al.

A marker for stromal stress and reporter for endoplasmic

Modulators of circadian rhythm and stromal signaling et al. 2002)

Coray 2009)

Holland 2005)

et al. 2007)

al. 2009)

2009)

2010)

al. 2010)

al. 2010)

Woolfenden

Vykhovanets

al. 2008), Robbins

Moriyama et al. 2005)

Hiseh 2005(Hsieh, Xie et al. 2005), Lyons 2006(Lyons, Lim et al. 2006), Iyer

2005(Iyer, Salazar et al. 2004; Iyer, Salazar et al. 2005)

2008(Vykhovanets, Shukla et

2011(Robbins and Zhao 2011)

Zhang 2009(Zhang, Lyons et

2009(Woolfenden, Zhu et al.

Geusz 2010(Geusz, Blakely et

Spiotto 2010(Spiotto, Banh et

Zumsteg 2010(Zumsteg, Strittmatter et al. 2010)

Inflammation Moriyama 2005(Moriyama,

Rehemtulla 2004(Rehemtulla, Taneja et al. 2004), Briat 2008(Briat and Vassaux 2008)

Lin 2005(Lin, Luo et al. 2005), Luo 2009(Luo and Wyss-

**Transgene Research focus Reference** 

p53-Luc Cell cycle, apoptosis

(SBE-Luc) TGFbeta/Smad signaling

(PSA-Luc) Hormone receptor activity

NFkB-Luc Signaling and inflammation

tumors

multiforme model

carcinogenesis

to tumors

reticulum

EL1-Luc/TAg Spontaneous pancreatic

Cre/Lox Luc Conditional glioblastoma

RipTag-IRES-Luc Pancreatic beta cell

able to regress.

Caspase cleavage sequence

Smad-responsive element

Inducible Nitric Oxide Synthase (FVB/N-Tg(iNOS-

Prostate Specific Antigen

Vascular Endothelial Growth

Mouse Period 1 (mPer1-luc)

X-box binding protein 1

(XBP1-Luc)

Luc)Xen)

Fig. 2. Bioluminescence imaging of luciferase activity in PSA-Luc transgenic mice. BLI following injection of luciferin in control (A), PSA-Luc transgenic males (B), and PSA-Luc transgenic female (C) mice at 10–12 weeks of age. Isolated prostate, epididymis and tails from 12-week-old PSA-Luc male mice were imaged following luciferin administration (D). (Hsieh, C. L., Z. Xie, et al. (2005). "A luciferase transgenic mouse model: visualization of prostate development and its androgen responsiveness in live animals." J Mol Endocrinol 35(2): 293-304. Reproduced by permission.)

The PSA-Luc animal is just one example of a transgenic mouse models available for BLI. Table 2 summarizes a list of luciferase transgenic mouse models that have been established over the last 10 years and used recently in preclinical studies of cancer. Many of these strains are now commercially available. Goldman et al use the Jackson Laboratory ODD-Luc transgenic mouse strain to construct a spontaneous tumor model with a built in reporter that can be used to localize tumors. The ODD-Luc transgenic mouse expresses HIF-1α oxygen-dependent degradation domain (ODD) fused to luciferase in all tissues, however, under hypoxic stress the ODD-Luc accumulates in hypoxic tissue and is readily observed by BLI. In the Goldman study, the ODD-Luc mice were crossed with Mouse Mammary Tumor Virus (MMTV) transgenic mice. MMTV can act as an insertional mutagen or induce transcription of nearby oncogenes post-insertion leading to the development malignant tumors in the mammary gland of infected mice. The MMTV transgenic mouse has been engineered to express the MMTV-LTR which predisposes the animal to develop multiple spontaneous mammary tumors (Taneja, Frazier et al. 2009). Since solid tumors often contain hypoxic centers, this group demonstrated that the ODD-Luc/MMTV transgenic mouse can be used to follow spontaneous tumor development, progression and response to treatment.

A

B

The PSA-Luc animal is just one example of a transgenic mouse models available for BLI. Table 2 summarizes a list of luciferase transgenic mouse models that have been established over the last 10 years and used recently in preclinical studies of cancer. Many of these strains are now commercially available. Goldman et al use the Jackson Laboratory ODD-Luc transgenic mouse strain to construct a spontaneous tumor model with a built in reporter that can be used to localize tumors. The ODD-Luc transgenic mouse expresses HIF-1α oxygen-dependent degradation domain (ODD) fused to luciferase in all tissues, however, under hypoxic stress the ODD-Luc accumulates in hypoxic tissue and is readily observed by BLI. In the Goldman study, the ODD-Luc mice were crossed with Mouse Mammary Tumor Virus (MMTV) transgenic mice. MMTV can act as an insertional mutagen or induce transcription of nearby oncogenes post-insertion leading to the development malignant tumors in the mammary gland of infected mice. The MMTV transgenic mouse has been engineered to express the MMTV-LTR which predisposes the animal to develop multiple spontaneous mammary tumors (Taneja, Frazier et al. 2009). Since solid tumors often contain hypoxic centers, this group demonstrated that the ODD-Luc/MMTV transgenic mouse can be used to follow spontaneous tumor development, progression and response to treatment.

Fig. 2. Bioluminescence imaging of luciferase activity in PSA-Luc transgenic mice. BLI following injection of luciferin in control (A), PSA-Luc transgenic males (B), and PSA-Luc transgenic female (C) mice at 10–12 weeks of age. Isolated prostate, epididymis and tails from 12-week-old PSA-Luc male mice were imaged following luciferin administration (D). (Hsieh, C. L., Z. Xie, et al. (2005). "A luciferase transgenic mouse model: visualization of prostate development and its androgen responsiveness in live animals." J Mol Endocrinol

35(2): 293-304. Reproduced by permission.)

They were able to use BL to image the growth and development of spontaneous tumors and showed that in response to treatment using doxorubicin and prednisone, these tumors were able to regress.


Bioluminescence Applications in Preclinical Oncology Research 147

Cao et al. 2002). Using topographical scanning it is now possible to construct a three dimensional image of the animal at the same time BL data is being collected. This combined imaging may help to provide better resolution and signal localization. The IVIS 200 system is able to create a 3 dimensional image of the animal where a scanning laser positioned in the horizontal plane is used to make a measurement of surface topography as shown in Figure 3B. This image is converted into a digital reconstruction of the animal that can be superimposed onto an animal atlas and used to localize the depth of signal as seen in Figure 3C. Newer BL imaging systems such as the Spectrum CT by Caliper are able to simultaneously create a Computed Tomography scan for the purpose of constructing a three dimensional image of the animal (Figure 3D). Multi-modal imaging can aid in localization, and an assessment of the depth of signal within body cavities with more precision than BLI

**Company System Method used for supercooling** 

Caliper LifeSciences IVIS Thermoelectric cooling Andor iXon Thermoelectric cooling

Berthold NightOWL Pelitier cooling Hamamatsu + VIM camera Intensified cooling Improvision Model C2400-47 Cryogenic cooling Roper Scientific ChemiPro Cryogenic cooling Biospace PhotoImager Intensified cooling Koday In vivo FX Thermoelectric cooling

Table 3. Commercially available BL imaging systems (Baert 2008)

A B C D

Sciences. A grayscale digital image is taken at the same time as photon capture, subsequently BL is superimposed onto the digital image (A). Surface topography is constructed using horizontal laser scanning (B), and used to create 3D rendering of the animal which can be overlain on an organ atlas (C). Figure C shows red dots where photon intensity is highest, indicating the depth and localization of the signal. Newer systems incorporate a CT scan with BL (D) in order to gain higher precision in signal localization.

(Reproduced by permission from Caliper Life Sciences, Hopkinton, MA, USA.)

Fig. 3. 2D and 3D detection of F-Luc labeled cells using the IVIS system from Caliper Life

alone.


Table 2. Luciferase based transgenic mouse models with applications in preclinical cancer research
