**5. Use of BLI in oncology research**

In oncology research, BL was first used in vitro and in vivo to assess cellular metabolism and indirectly to measure viability in an experiment known as the ATP assay. In this assay, luciferin and luciferase are added to media and in the presence of ATP, luciferin is oxidized giving off light that can be measured using a luminometer or a CCD camera. These assays are a direct measure of cell viability and can be used to assess cell proliferation and cytotoxicity in vitro (Garewal, Ahmann et al. 1986; Kuzmits, Aiginger et al. 1986; Kuzmits, Rumpold et al. 1986; Ahmann, Garewal et al. 1987; Sevin, Peng et al. 1988; Petru, Sevin et al. 1990; Crouch, Kozlowski et al. 1993). Using the ATP assay Garewal et al were able to distinguish between cytostatic and cytotoxic effects of therapeutics on a colon cancer cell line in vitro (Garewal, Ahmann et al. 1986).

In the late 80s and early 90's Mueller-Klieser and Walenta et al began mapping metabolites in excised tumor tissues using similar principles to the ATP assay. Their method allowed for the assessment of glucose, lactate, and ATP distributions in sections of tumors and normal tissue from cryobiopsies. Briefly, the procedure involves application of a solution containing gelatin and enzymes linked to luciferase. Upon tissue thawing, the enzyme solution diffuses into the tissue section initiating the BL reaction wherever the substrate of interest is found. The photons emitted are visualized in tumor sections using a CCD equipped microscope (Mueller-Klieser, Walenta et al. 1988; Mueller-Klieser, Kroeger et al. 1991; Walenta, Schroeder et al. 2002). Figure 4 shows a bright-field image (A) and BLI of ATP (B) from a cryosection of melanoma from the Syrian golden hamster. The BLI clearly indicates high concentrations of ATP in viable cell regions of the periphery. Furthermore, the studies show that high levels of glucose are found in the tumor periphery while necrotic tumor centers exhibited high lactate levels (Walenta, Dellian et al. 1992).

Bioluminescence Applications in Preclinical Oncology Research 151

The non-invasive utility of BLI as a small-animal imaging modality has led to the development of a wide range of luciferase positive orthotopic and metastatic animal models. Orthotopic models for breast cancer (Garcia, Jackson et al. 2008; Shan, Wang et al. 2008; Kalra, Warburton et al. 2009; Kalra, Anantha et al. 2011), bladder cancer (Mugabe, Matsui et al. 2011; van der Horst, van Asten et al. 2011), hepatocellular carcinoma (Frampas, Maurel et al. 2011), ovarian cancer (Cordero, Kwon et al. 2010; Bevis, McNally et al. 2011), head and neck SCC (Sano, Matsumoto et al. 2011), multiple myeloma (Runnels, Carlson et al. 2011), pancreatic cancer (Angst, Chen et al. 2010; McNally, Welch et al. 2010; Muniz, Barnes et al. 2011), glioma (Prasad, Sottero et al. 2011), lung cancer(Madero-Visbal, Colon et al. 2010; Li, Torossian et al. 2011; Yan, Xiao et al. 2011), prostate cancer (Svensson, Haverkamp et al. 2011), mesothelioma (Feng, Zhang et al. 2011), neuroblastoma (Teitz, Stanke et al. 2011; Tivnan, Tracey et al. 2011), rectal cancer (Huerta, Gao et al. 2011), renal cancer (Karam, Mason et al. 2003), sarcoma (Vikis, Jackson et al. 2010) have been established in vivo. Furthermore, several of these models have been used to develop systemic disease. For example, Mishra et al use intracardiac and intratibial inoculation of luciferase positive prostate cancer cells to investigate the effect of inhibiting TGFβ on osteoblastic tumor growth and incidence in vivo (Mishra, Tang et al. 2011). As seen below, our group used a luciferase positive human breast cancer cell line (LCC6-Luc) to establish orthotopic disease via mammary fatpad injections, ascitic disease via intraperitoneal injection and metastatic

**5.1 Capturing minimal disease as well as quantifying tumor development and growth**  Luciferase expressing cells can be used in vivo to monitor tumor growth. In an animal model, it is possible to observe small numbers of luciferase positive cells following luciferin administration. Indeed, post-inoculation, luciferase activity can be detected allowing for the confirmation of cell injection. Because small numbers of cells are readily detected, quantitative measurements of disease burden can be done earlier and for the identification of metastatic spread. A study done in our laboratory used LCC6-Luc cells to inoculate animals orthotopically, intracardiac, or intraperitoneal, to establish mammary tumors, systemic disease and ascities disease respectively. The results are summarized in Figure 6. This study demonstrated that the growth of orthotopic, systemic, and ascities disease can be monitored from day zero upon tumor cell inoculation, through to day 28 where there is established disease. The BLI data was quantified and used to create growth curves for each model. Further, the information provided in Figure 1 was used to estimate the number of cells detected at day 7, 14, 21 and 28. These data, in turn, could be used to generate a tumor

As suggested by the above example, a major advantage of using BLI is the sensitivity of capturing photon emissions from small numbers of cells and monitoring the onset of disease, tumor growth, primary tumor dynamics and progression to metastatsis. Using BLI it is possible to quantify the kinetics of tumor growth since photon emissions increase in proportion to the number of cells and thus disease burden. Light measurements can be made from whole body scans or from a selected region of interest and are most commonly quantified as total photon counts (photons/s). One of the first in vivo experiments performed using BLI of luciferase labeled cells was done by Edinger et al in 1999. This group

disease via intracardiac inoculation (Kalra, Anantha et al. 2011).

growth curve (Kalra, Anantha et al. 2011).

Fig. 4. Mapping ATP in melanoma of the Syrian Hamster using BLI. Cryosection stained with hematoxylin and eosin (A) shows necrosis in the center of the tumor, Colour-coded intensity image of bioluminescence (B) illustrates the local distrubiton of ATP concentrations. (Walenta, S., M. Dellian, et al. (1992). "Pixel-to-pixel correlation between images of absolute ATP concentrations and blood flow in tumors." Br J Cancer 66(6): 1099- 1102. Reproduced by permission.)

The metabolic switch to aerobic glycolysis and enhanced lactate production is characteristic for aggressive tumor cells and a factor for tumor response and treatment outcome. Thus, BL mapping of metabolites can be used as an early marker for treatment response. Broggini-Tenzer et al use BL metabolite mapping strategies in mice carrying tumor xenografts derived from A549 lung cancer cells to show that metabolite levels are influenced by treatment with the microtubule stabilizing agent patupilone, ionizing radiation or a combination of the two modalities (Broggini-Tenzer, Vuong et al. 2011). Their results are shown in Figure 5. The BLI of tumor sections indicated that lactate levels were significantly reduced and glucose levels drastically increased in treated tumors compare to the untreated tumors. However, ATP levels did not change significantly with any of the treatments used.

Fig. 5. Distribution of metabolites within lung tumor sections after treatment. Representative tumors are shown for each treatment group and consecutive sections were stained for ATP, lactate and glucose. Treatment with patupilone or irradiation exhibit decreased ATP levels and Lactate levels but increased glucose levels within the tumor core. (Broggini-Tenzer, A., V. Vuong, et al. (2011). "Metabolism of tumors under treatment: mapping of metabolites with quantitative bioluminescence." Radiother Oncol 99(3): 398-403.Reproduced by permission.)

A B Fig. 4. Mapping ATP in melanoma of the Syrian Hamster using BLI. Cryosection stained with hematoxylin and eosin (A) shows necrosis in the center of the tumor, Colour-coded

concentrations. (Walenta, S., M. Dellian, et al. (1992). "Pixel-to-pixel correlation between images of absolute ATP concentrations and blood flow in tumors." Br J Cancer 66(6): 1099-

The metabolic switch to aerobic glycolysis and enhanced lactate production is characteristic for aggressive tumor cells and a factor for tumor response and treatment outcome. Thus, BL mapping of metabolites can be used as an early marker for treatment response. Broggini-Tenzer et al use BL metabolite mapping strategies in mice carrying tumor xenografts derived from A549 lung cancer cells to show that metabolite levels are influenced by treatment with the microtubule stabilizing agent patupilone, ionizing radiation or a combination of the two modalities (Broggini-Tenzer, Vuong et al. 2011). Their results are shown in Figure 5. The BLI of tumor sections indicated that lactate levels were significantly reduced and glucose levels drastically increased in treated tumors compare to the untreated tumors. However, ATP levels did not change significantly with any of the treatments used.

Fig. 5. Distribution of metabolites within lung tumor sections after treatment. Representative tumors are shown for each treatment group and consecutive sections were stained for ATP, lactate and glucose. Treatment with patupilone or irradiation exhibit decreased ATP levels and Lactate levels but increased glucose levels within the tumor core. (Broggini-Tenzer, A., V. Vuong, et al. (2011). "Metabolism of tumors under treatment: mapping of metabolites with quantitative bioluminescence." Radiother Oncol 99(3): 398-403.Reproduced by

intensity image of bioluminescence (B) illustrates the local distrubiton of ATP

1102. Reproduced by permission.)

permission.)

The non-invasive utility of BLI as a small-animal imaging modality has led to the development of a wide range of luciferase positive orthotopic and metastatic animal models. Orthotopic models for breast cancer (Garcia, Jackson et al. 2008; Shan, Wang et al. 2008; Kalra, Warburton et al. 2009; Kalra, Anantha et al. 2011), bladder cancer (Mugabe, Matsui et al. 2011; van der Horst, van Asten et al. 2011), hepatocellular carcinoma (Frampas, Maurel et al. 2011), ovarian cancer (Cordero, Kwon et al. 2010; Bevis, McNally et al. 2011), head and neck SCC (Sano, Matsumoto et al. 2011), multiple myeloma (Runnels, Carlson et al. 2011), pancreatic cancer (Angst, Chen et al. 2010; McNally, Welch et al. 2010; Muniz, Barnes et al. 2011), glioma (Prasad, Sottero et al. 2011), lung cancer(Madero-Visbal, Colon et al. 2010; Li, Torossian et al. 2011; Yan, Xiao et al. 2011), prostate cancer (Svensson, Haverkamp et al. 2011), mesothelioma (Feng, Zhang et al. 2011), neuroblastoma (Teitz, Stanke et al. 2011; Tivnan, Tracey et al. 2011), rectal cancer (Huerta, Gao et al. 2011), renal cancer (Karam, Mason et al. 2003), sarcoma (Vikis, Jackson et al. 2010) have been established in vivo. Furthermore, several of these models have been used to develop systemic disease. For example, Mishra et al use intracardiac and intratibial inoculation of luciferase positive prostate cancer cells to investigate the effect of inhibiting TGFβ on osteoblastic tumor growth and incidence in vivo (Mishra, Tang et al. 2011). As seen below, our group used a luciferase positive human breast cancer cell line (LCC6-Luc) to establish orthotopic disease via mammary fatpad injections, ascitic disease via intraperitoneal injection and metastatic disease via intracardiac inoculation (Kalra, Anantha et al. 2011).

#### **5.1 Capturing minimal disease as well as quantifying tumor development and growth**

Luciferase expressing cells can be used in vivo to monitor tumor growth. In an animal model, it is possible to observe small numbers of luciferase positive cells following luciferin administration. Indeed, post-inoculation, luciferase activity can be detected allowing for the confirmation of cell injection. Because small numbers of cells are readily detected, quantitative measurements of disease burden can be done earlier and for the identification of metastatic spread. A study done in our laboratory used LCC6-Luc cells to inoculate animals orthotopically, intracardiac, or intraperitoneal, to establish mammary tumors, systemic disease and ascities disease respectively. The results are summarized in Figure 6. This study demonstrated that the growth of orthotopic, systemic, and ascities disease can be monitored from day zero upon tumor cell inoculation, through to day 28 where there is established disease. The BLI data was quantified and used to create growth curves for each model. Further, the information provided in Figure 1 was used to estimate the number of cells detected at day 7, 14, 21 and 28. These data, in turn, could be used to generate a tumor growth curve (Kalra, Anantha et al. 2011).

As suggested by the above example, a major advantage of using BLI is the sensitivity of capturing photon emissions from small numbers of cells and monitoring the onset of disease, tumor growth, primary tumor dynamics and progression to metastatsis. Using BLI it is possible to quantify the kinetics of tumor growth since photon emissions increase in proportion to the number of cells and thus disease burden. Light measurements can be made from whole body scans or from a selected region of interest and are most commonly quantified as total photon counts (photons/s). One of the first in vivo experiments performed using BLI of luciferase labeled cells was done by Edinger et al in 1999. This group

Bioluminescence Applications in Preclinical Oncology Research 153

More recently in 2011, van der Horst et al used a re-engineered firefly luciferase (Luciferase 2) in which a transcription-factor binding sites that compromised luciferase expression was removed. A Luciferase 2-positive human transitional carcinoma cell line (UM-UC-3-Luc) was used to inoculate either the bladder of mice to produce an orthotopic model with systemic metastases or in the left cardiac ventricle to develop a model that simulates bone metastasis. This group was able to detect 100 cells three hours after subcutaneous inoculation. They were also able to detect micrometastases from both the orthotopic and

In 2002, Bhaumik et al show that D-luciferin (the substrate for F-Luc) does not serve as a substrate for Renilla Luciferase (R-Luc), and coelenterazine (the substrate for R-Luc) does not serve as a substrate for F-Luc in cell culture or in living mice. This group made stable transfections of a rat glioma cell line (C6) with R-Luc or F-Luc. Mice were inoculated with C6-R-Luc in the left forearm, and C6-F-Luc in the right forearm. Once tumors had established, animals were subject to BLI using D-luciferin or coelenterazine. As shown in Figure 7, D-luciferin delivery was associated with photons emitted from the tumor cells in the right forearm, while coeleterazine was associated with photons emitted from the left forearm. Elegantly, Bhaumik et al were able to demonstrate that both R-Luc and F-Luc expression can be imaged in the same living mouse. This pivotal finding adds an extra layer of complexity to the BLI modality in that different luciferins can be used to track at least two separate 1) molecular events, 2) cell populations such as stem cells versus tumor cells, 3) gene therapy vectors, or 4) endogenous genes through the use of two reporter luciferase

A B Fig. 7. BLI of F-Luc and R-Luc activity in the same animal. Both C6-F-Luc (A) and C6-R-Luc (B) cells were implanted subcutaneously at the right or left forearm sites respectively in the

bioluminescence from site A and minimal signal from site B. Injection of coelenterazine via tail-vein in the mouse in Figure 3B exhibit bioluminescence from site B but minimal signal from site A. (Bhaumik, S. and S. S. Gambhir (2002). "Optical imaging of Renilla luciferase reporter gene expression in living mice." 99 (1): 377-382. Copyright (2002) National

Indeed using Bhaumik's findings, Wang et al separately labeled murine breast cancer cells (4T1) with an R-Luc-monomeric red fluorescence protein (R-Luc-mRFP) reporter vector and mesenchymal stem cells (MSC) with a F-Luc-enhanced green fluorescence protein (F-Luc-

same mouse. Injection of D-luciferin via tail-vein in the mouse in figure 4A shows

Academy of Sciences, U.S.A. Reproduced by permission.)

metastatic tumors (van der Horst, van Asten et al. 2011).

genes (Bhaumik and Gambhir 2002).

used a Luciferase positive human cervical carcinoma cancer cell line (HeLa-Luc) to inoculate animals via subcutaneous, intraperitoneal and intravenous injection. They were able to visualize cells immediately following inoculation using all of the injection routes. According to this study, 1 × 103 cells could be detected in the peritoneal cavity, 1 × 104 at subcutaneous sites, and 1 × 106 circulating cells following injection (Edinger, Sweeney et al. 1999).

Fig. 6. Using Luciferase positive to establish orthotopic, systemic and ascitic tumors in animals. LCC6WT-Luc cells were inoculated orthotopically (A & B), via intracardiac injection (C & D), or intraperitoneally (E & F). BLI was used to monitor tumor growth. Images shown were acquired on days 0, 1, 7, 14, 21, and 28. Photon emissions were measured using whole body scans for each animal (representative images shown in A, C and E). BL data was quantified to generate growth curves (B, D and F). The inset graphs represent BLI data as it correlates to the number of cells. (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 from the author.)

In 2003 Jenkins et al developed a luciferase positive prostate cancer, lung cancer and colon cancer model to study tumor growth in vivo. Bioluminescent PC-3M-Luc-C6 human prostate cancer cells were implanted subcutaneously into mice and were monitored for tumor growth using BLI. They show that BLI data correlated to standard external caliper measurements of tumor volume, but BLI permitted earlier detection of tumor cells. In the lung colonization cancer model, bioluminescent A549-Luc-C8 human lung cancer cells were injected intravenously and lung metastases were successfully monitored in vivo by whole animal imaging. Bioluminescent HT-29-Luc-D6 human colon cancer cells implanted subcutaneously produced metastases to lung and lymph nodes. Both primary tumors and micrometastases were detected by BLI in vivo (Jenkins, Oei et al. 2003).

used a Luciferase positive human cervical carcinoma cancer cell line (HeLa-Luc) to inoculate animals via subcutaneous, intraperitoneal and intravenous injection. They were able to visualize cells immediately following inoculation using all of the injection routes. According to this study, 1 × 103 cells could be detected in the peritoneal cavity, 1 × 104 at subcutaneous

sites, and 1 × 106 circulating cells following injection (Edinger, Sweeney et al. 1999).

Fig. 6. Using Luciferase positive to establish orthotopic, systemic and ascitic tumors in animals. LCC6WT-Luc cells were inoculated orthotopically (A & B), via intracardiac injection (C & D), or intraperitoneally (E & F). BLI was used to monitor tumor growth. Images shown were acquired on days 0, 1, 7, 14, 21, and 28. Photon emissions were measured using whole body scans for each animal (representative images shown in A, C and E). BL data was quantified to generate growth curves (B, D and F). The inset graphs represent BLI data as it correlates to the number of cells. (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

In 2003 Jenkins et al developed a luciferase positive prostate cancer, lung cancer and colon cancer model to study tumor growth in vivo. Bioluminescent PC-3M-Luc-C6 human prostate cancer cells were implanted subcutaneously into mice and were monitored for tumor growth using BLI. They show that BLI data correlated to standard external caliper measurements of tumor volume, but BLI permitted earlier detection of tumor cells. In the lung colonization cancer model, bioluminescent A549-Luc-C8 human lung cancer cells were injected intravenously and lung metastases were successfully monitored in vivo by whole animal imaging. Bioluminescent HT-29-Luc-D6 human colon cancer cells implanted subcutaneously produced metastases to lung and lymph nodes. Both primary tumors and

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

micrometastases were detected by BLI in vivo (Jenkins, Oei et al. 2003).

More recently in 2011, van der Horst et al used a re-engineered firefly luciferase (Luciferase 2) in which a transcription-factor binding sites that compromised luciferase expression was removed. A Luciferase 2-positive human transitional carcinoma cell line (UM-UC-3-Luc) was used to inoculate either the bladder of mice to produce an orthotopic model with systemic metastases or in the left cardiac ventricle to develop a model that simulates bone metastasis. This group was able to detect 100 cells three hours after subcutaneous inoculation. They were also able to detect micrometastases from both the orthotopic and metastatic tumors (van der Horst, van Asten et al. 2011).

In 2002, Bhaumik et al show that D-luciferin (the substrate for F-Luc) does not serve as a substrate for Renilla Luciferase (R-Luc), and coelenterazine (the substrate for R-Luc) does not serve as a substrate for F-Luc in cell culture or in living mice. This group made stable transfections of a rat glioma cell line (C6) with R-Luc or F-Luc. Mice were inoculated with C6-R-Luc in the left forearm, and C6-F-Luc in the right forearm. Once tumors had established, animals were subject to BLI using D-luciferin or coelenterazine. As shown in Figure 7, D-luciferin delivery was associated with photons emitted from the tumor cells in the right forearm, while coeleterazine was associated with photons emitted from the left forearm. Elegantly, Bhaumik et al were able to demonstrate that both R-Luc and F-Luc expression can be imaged in the same living mouse. This pivotal finding adds an extra layer of complexity to the BLI modality in that different luciferins can be used to track at least two separate 1) molecular events, 2) cell populations such as stem cells versus tumor cells, 3) gene therapy vectors, or 4) endogenous genes through the use of two reporter luciferase genes (Bhaumik and Gambhir 2002).

Fig. 7. BLI of F-Luc and R-Luc activity in the same animal. Both C6-F-Luc (A) and C6-R-Luc (B) cells were implanted subcutaneously at the right or left forearm sites respectively in the same mouse. Injection of D-luciferin via tail-vein in the mouse in figure 4A shows bioluminescence from site A and minimal signal from site B. Injection of coelenterazine via tail-vein in the mouse in Figure 3B exhibit bioluminescence from site B but minimal signal from site A. (Bhaumik, S. and S. S. Gambhir (2002). "Optical imaging of Renilla luciferase reporter gene expression in living mice." 99 (1): 377-382. Copyright (2002) National Academy of Sciences, U.S.A. Reproduced by permission.)

Indeed using Bhaumik's findings, Wang et al separately labeled murine breast cancer cells (4T1) with an R-Luc-monomeric red fluorescence protein (R-Luc-mRFP) reporter vector and mesenchymal stem cells (MSC) with a F-Luc-enhanced green fluorescence protein (F-Luc-

Bioluminescence Applications in Preclinical Oncology Research 155

to determine whether the luciferase positive model would allow for monitoring of response to therapeutic interventions. Animals were treated with paclitaxel or irradiated, and tumor burden was monitored using BLI. They noted that tumors responded to paclitaxel or radiation as shown by decreased tumor BL which also correlated to improved overall survival (Li, Torossian et al. 2011). Mugabe et al, use BLI to determine if a mucoadhesive nanoparticulate form of docetaxel is able to improve treatment of a bladder cancer by increasing the dwell time and uptake of the intravesical drug (Mugabe, Matsui et al. 2011). In 2009, Graeser et al use BLI to show that a liposomal formulation of gemcitabine has improved anti-tumor and anti-metastatic effects in an orthotopic model of pancreatic cancer when compared to the free drug (Graeser, Bornmann et al. 2009). As most agents are used as part of combination regimens, BLI is an ideal technique to study combination effects against single agent therapies and to elucidate combinatorial ratios and scheduling, as these experiments require multiple study arms and large numbers of animals. Prasad et al show that in a luciferase positive glioma model, combining a cytostatic agent (PI3K/mTOR dual inhibitor (XL765)) with a clinically relevant agent, temozolomide (TMZ), resulted in an additive reduction in tumor BL compared with control. The BLI data correlated to improvement in median survival time in the combination treated group (Prasad, Sottero et al. 2011). Finally, using a novel approach to study pharmacodynamics, Pensel et al use two imaging modalities to study probe accumulation at the site of tumor tissue. This group used a luciferase positive human leukemia model (HL-60-Luc) and a radiolabeled probe (spermine), imaged with BLI and SPECT respectively, to demonstrate that the spermine

In addition to monitoring tumor initiation and growth as indicated above, BLI has been used in a variety of mechanistic studies. For example, in 2002 Shuetz et al were able to track transcription of luciferase reporter genes in an in vivo model of liver cancer before and after treatment (Schuetz, Lan et al. 2002). In 2003 Luker et al used a ubiquitin luciferase reporter to follow protesomal function in vivo before and after treatment with proteosome inhibitors (Luker, Pica et al. 2003). In oncology research it is now possible to devise reporter strategies to assess key cancer processes such as dysregulated signaling, induction of apoptosis and

Imaging apoptosis in vivo using a non-invasive modality would be a valuable method to evaluate drugs that induce programmed cell death. To this end, Laxman et al constructed an apoptosis biosensor by fusing the estrogen regulatory (ER) domain to F-Luc. The ER domain is able to silence the enzymatic activity of luciferase. The construct was further engineered so that the luciferase protein was flanked by the protease cleavage site for caspase-3. This cleavage site consists of aspartic acid (D), glutamic acid (E), valine (V), and aspartic acid (D) and is known as DEVD. If the DEVD site is cleaved by caspase-3, luciferase would be released from the construct, and the silencing effect from ER would be ablated. Stable human glioma cells line expressing this luciferase construct were generated and inoculated into animals subcutaneously. Animals were treated with tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL), which induces apoptosis. With activation of caspase-3

conjugate accumulates in tumor cells (Pesnel, Guminski et al. 2011).

**5.2 Investigating key cancer processes in vivo** 

angiogeneis in vivo.

**5.2.1 Imaging apoptosis** 

eGFP) reporter vector in order to study how MSC traffic and differentiate in either subcutaneous or metastatic animal models. Wang et al were successfully able to monitor tumor growth by R-Luc BLI and the MSC's by F-Luc BLI in the same animal (Wang, Cao et al. 2009).

As already indicated, F-Luc requires ATP in order to produce light, thus only metabolically active and oxygen rich cells contribute to the signal observed in BLI. A decrease in signal intensity occurs as cells undergo apoptosis or necrosis. In one of our own studies, we used the highly aggressive breast cancer cell line MDA MB 435/LCC6 to make a BL orthotopic mouse model. This cell line is known to rapidly develop tumors with necrotic cores. Using BLI it was noted that 28 days post tumor inoculation, the center of the tumor no longer emitted BL photons suggestive of a metabolically inactive tumor core. Dead or necrotic regions within a tumor, would still contribute to its volume, therefore traditional caliper measurements would have provided inaccurate results.

Fig. 8. Orthotopic LCC6-Luc tumors have necrotic cores that can be visualized using BLI. LCC6-Luc cells were inoculated into the mammary fat pad of female mice. Tumors were monitored using BLI. On day 28 post tumor cell inoculation, non-luminescing regions within the tumor was visualized after BLI suggestive of necrotic centers. (Kalra, J., C. Warburton, et al. (2009). "QLT0267, a small molecule inhibitor targeting integrin-linked kinase (ILK), and docetaxel can combine to produce synergistic interactions linked to enhanced cytotoxicity, reductions in P-AKT levels, altered F-actin architecture and improved treatment outcomes in an orthotopic breast cancer model." Breast Cancer Res 11(3): R25. Reproduced by permission.)

Since only metabolically active cells may produce BL, BLI can be used to indicate a positive drug response. BLI can be used to measure cell death with treatment using cytotoxic agents or even reduced proliferation post treatment with cytostatic agents. Many studies have been published to date describing the use of BLI to determine the efficacy of a variety of drugs currently being used in clinic and to look for valuable new drugs in the development pipeline. Because BLI is able to capture minimal disease, treatment success and cure of animals can be determined at earlier stages of, at metastasis and to monitor relapse before any clinical signs of disease are detectable. Further, since animals are monitored noninvasively, serial sacrifice of groups of animals at intermediate time-points is not necessary. This saves on the number of animals used per study and also means that multiple parameters of drug efficacy and dosing can be studied simultaneously. Li et al used luciferase-expressing A549 cancer cells injected into the mediastinum of athymic nude mice

eGFP) reporter vector in order to study how MSC traffic and differentiate in either subcutaneous or metastatic animal models. Wang et al were successfully able to monitor tumor growth by R-Luc BLI and the MSC's by F-Luc BLI in the same animal (Wang, Cao

As already indicated, F-Luc requires ATP in order to produce light, thus only metabolically active and oxygen rich cells contribute to the signal observed in BLI. A decrease in signal intensity occurs as cells undergo apoptosis or necrosis. In one of our own studies, we used the highly aggressive breast cancer cell line MDA MB 435/LCC6 to make a BL orthotopic mouse model. This cell line is known to rapidly develop tumors with necrotic cores. Using BLI it was noted that 28 days post tumor inoculation, the center of the tumor no longer emitted BL photons suggestive of a metabolically inactive tumor core. Dead or necrotic regions within a tumor, would still contribute to its volume, therefore traditional caliper

Fig. 8. Orthotopic LCC6-Luc tumors have necrotic cores that can be visualized using BLI. LCC6-Luc cells were inoculated into the mammary fat pad of female mice. Tumors were monitored using BLI. On day 28 post tumor cell inoculation, non-luminescing regions within the tumor was visualized after BLI suggestive of necrotic centers. (Kalra, J., C. Warburton, et al. (2009). "QLT0267, a small molecule inhibitor targeting integrin-linked kinase (ILK), and docetaxel can combine to produce synergistic interactions linked to enhanced cytotoxicity, reductions in P-AKT levels, altered F-actin architecture and improved treatment outcomes in an orthotopic breast cancer model." Breast Cancer Res

Since only metabolically active cells may produce BL, BLI can be used to indicate a positive drug response. BLI can be used to measure cell death with treatment using cytotoxic agents or even reduced proliferation post treatment with cytostatic agents. Many studies have been published to date describing the use of BLI to determine the efficacy of a variety of drugs currently being used in clinic and to look for valuable new drugs in the development pipeline. Because BLI is able to capture minimal disease, treatment success and cure of animals can be determined at earlier stages of, at metastasis and to monitor relapse before any clinical signs of disease are detectable. Further, since animals are monitored noninvasively, serial sacrifice of groups of animals at intermediate time-points is not necessary. This saves on the number of animals used per study and also means that multiple parameters of drug efficacy and dosing can be studied simultaneously. Li et al used luciferase-expressing A549 cancer cells injected into the mediastinum of athymic nude mice

measurements would have provided inaccurate results.

11(3): R25. Reproduced by permission.)

et al. 2009).

to determine whether the luciferase positive model would allow for monitoring of response to therapeutic interventions. Animals were treated with paclitaxel or irradiated, and tumor burden was monitored using BLI. They noted that tumors responded to paclitaxel or radiation as shown by decreased tumor BL which also correlated to improved overall survival (Li, Torossian et al. 2011). Mugabe et al, use BLI to determine if a mucoadhesive nanoparticulate form of docetaxel is able to improve treatment of a bladder cancer by increasing the dwell time and uptake of the intravesical drug (Mugabe, Matsui et al. 2011). In 2009, Graeser et al use BLI to show that a liposomal formulation of gemcitabine has improved anti-tumor and anti-metastatic effects in an orthotopic model of pancreatic cancer when compared to the free drug (Graeser, Bornmann et al. 2009). As most agents are used as part of combination regimens, BLI is an ideal technique to study combination effects against single agent therapies and to elucidate combinatorial ratios and scheduling, as these experiments require multiple study arms and large numbers of animals. Prasad et al show that in a luciferase positive glioma model, combining a cytostatic agent (PI3K/mTOR dual inhibitor (XL765)) with a clinically relevant agent, temozolomide (TMZ), resulted in an additive reduction in tumor BL compared with control. The BLI data correlated to improvement in median survival time in the combination treated group (Prasad, Sottero et al. 2011). Finally, using a novel approach to study pharmacodynamics, Pensel et al use two imaging modalities to study probe accumulation at the site of tumor tissue. This group used a luciferase positive human leukemia model (HL-60-Luc) and a radiolabeled probe (spermine), imaged with BLI and SPECT respectively, to demonstrate that the spermine conjugate accumulates in tumor cells (Pesnel, Guminski et al. 2011).

#### **5.2 Investigating key cancer processes in vivo**

In addition to monitoring tumor initiation and growth as indicated above, BLI has been used in a variety of mechanistic studies. For example, in 2002 Shuetz et al were able to track transcription of luciferase reporter genes in an in vivo model of liver cancer before and after treatment (Schuetz, Lan et al. 2002). In 2003 Luker et al used a ubiquitin luciferase reporter to follow protesomal function in vivo before and after treatment with proteosome inhibitors (Luker, Pica et al. 2003). In oncology research it is now possible to devise reporter strategies to assess key cancer processes such as dysregulated signaling, induction of apoptosis and angiogeneis in vivo.

#### **5.2.1 Imaging apoptosis**

Imaging apoptosis in vivo using a non-invasive modality would be a valuable method to evaluate drugs that induce programmed cell death. To this end, Laxman et al constructed an apoptosis biosensor by fusing the estrogen regulatory (ER) domain to F-Luc. The ER domain is able to silence the enzymatic activity of luciferase. The construct was further engineered so that the luciferase protein was flanked by the protease cleavage site for caspase-3. This cleavage site consists of aspartic acid (D), glutamic acid (E), valine (V), and aspartic acid (D) and is known as DEVD. If the DEVD site is cleaved by caspase-3, luciferase would be released from the construct, and the silencing effect from ER would be ablated. Stable human glioma cells line expressing this luciferase construct were generated and inoculated into animals subcutaneously. Animals were treated with tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL), which induces apoptosis. With activation of caspase-3

Bioluminescence Applications in Preclinical Oncology Research 157

2 (VEGFR2) promoter that drives F-Luc expression. This mouse model can be used to monitor angiogenesis induced by tumors. Angst et al sought to investigate pancreatic cancer angiogenesis and thus employed the VEGFR2-Luc mouse. After orthotopic inoculation of pancreatic cells, light emission corresponding to VEGFR activity began at day 4, which this group suggests is likely due to wound healing, and continued throughout the experimental period during tumor growth suggesting angiogenesis was occurring. The BL results were confirmed using immunohistochemical staining for CD31 (Angst, Chen et al. 2010). In 2007, Faley et al generated a transgenic reporter mouse, VEGF-GFP/Luc, in which an enhanced green fluorescent protein-luciferase fusion protein is expressed under the control of a human VEGF-A promoter. The VEGF-GFP/Luc animals exhibited intense BL throughout the body at 1 week of age, but the signals declined as the mice grew so that the adult VEGF-GFP/Luc mouse showed BL only in areas undergoing active wound healing. However, in VEGF-GFP/Luc/MMTV mice, BL is observed in spontaneous tumors indicative of active

In order to have a mechanistic understanding of tumor biology and response to therapy, oncology research focuses on molecular alterations in the tumor or microenvironment. Under many circumstances up-regulation of oncogenes results in changes in protein–protein interactions, alterations in kinase activity and associated changes in important signalling pathways that promote tumour cell survival and proliferation. Much work has been accomplished to study these signalling cascades in vitro and in ex vivo tissue samples and as a result many therapies have been developed to target these dysregulated pathways. For these reasons there has been a great deal of interest in developing methods to visualize

Three general methods are currently available for imaging protein-protein interactions in living subjects using reporter genes: a modified mammalian two-hybrid system, a bioluminescence resonance energy transfer (BRET) system, and split reporter protein complementation and reconstitution strategies, these methods were reviewed by Massoud et al in 2007 (Massoud, Paulmurugan et al. 2007). Paulmurgan developed the split reporter system in vivo using very strongly interacting proteins MyoD and Id (Paulmurugan, Umezawa et al. 2002). In 2004 this same group used split synthetic R-Luc protein to evaluate heterodimerization of FRB and FKBP12 mediated by rapamycin. The rapamycin-mediated dimerization of FRB and FKBP12 was studied in living mice by locating, quantifying, and timing the R-Luc BL. Their work demonstrates that the split reporter system can be used to screen small molecule drugs that impact protein-protein interactions in living animals

It is also possible to use BLI for the evaluation of enzymatic activity such as kinase activity, in vivo. Khan et al established a luciferase-based reporter to image EGFR kinase activity in an in vivo model of squamous cell carcinoma (SCC). The EGFR Kinase reporter (EKR) is a multidomain chimeric reporter where BL can be used as a marker for EGFR kinase activity. The reporter is phosphorylated in the presence of active EGFR which interferes with luciferase activity, if the substrate is not phosphorylated BL is available for imaging. This reporter can therefore be used as an indicator for EGFR inhibition. Khan et al demonstrated

angiogenesis (Faley, Takahashi et al. 2007).

molecular changes in live animals.

(Paulmurugan, Massoud et al. 2004).

**5.2.3 Imaging Protein – Protein interactions and cell signalling** 

the DEVD sites were cleaved, luciferase was able to fold appropriately and upon exposure to luciferin, BL photons were produced. Therefore, apoptosis was successfully imaged noninvasively using BLI (Laxman, Hall et al. 2002). Using another methodology, Niers et al engineered the naturally secreted G-Luc so that it is separated by the DEVD sequence. They showed that this fusion protein was retained in the cytoplasm of transfected cells in an inactive form. Upon induction of apoptosis, the DEVD peptide was cleaved in response to caspase-3 activation, freeing G-Luc, which then entered the secretory pathway where it was folded properly and released from the cells. The G-Luc can be detected in the conditioned medium in culture or in blood from live animals (Niers, Kerami et al. 2011). Scabini et al 2011 use a similar approach however in this case a formulated Z-DEVD-aminoluciferin is delivered intraperiotneal to mice carrying human colon cancer or human glioblastoma cell lines engineered to express luciferase. Upon induction of apoptosis Z-DEVD-aminoluciferin is cleaved by caspase 3/7 releasing aminoluciferin that is now free to react with luciferase to generate measurable BL. This group was able to show that after camptothecin and temozolomide treatment of xenograft mouse models of colon cancer and glioblastoma respectively, the treated mice showed higher induction of Z-DEVD-aminoluciferin luminescent signal when compared to the untreated group. Combining D-luciferin that measures the total tumor burden, with Z-DEVD-aminoluciferin that assesses apoptosis induction via caspase activation, they were able to relate inhibition of tumor growth with induction of apoptosis after treatment in the same animal over time (Scabini, Stellari et al. 2011). Hickson et al use the same methodology in a luciferase positive ovarian cancer and breast cancer model. In these experiments, tumor cells were inoculated and allowed to establish, subsequently animals were treated with docetaxel. Animals were injected with the Z-DEVD-aminoluciferin before BL images were acquired. This group shows that more light was detected in the docetaxel-treated group compared with the untreated group (Hickson, Ackler et al. 2010).

#### **5.2.2 Imaging tumor hypoxia and angiogenesis**

Oxygen is needed for proper cellular metabolism, thus hypoxia, which is common in proliferating cancers, can significantly alter tumor biology on a molecular level. Monitoring hypoxia in vivo can provide important information on tumor biology and response to treatment. The transcription factor Hypoxia-inducing factor 1 (HIF1), is induced under conditions of hypoxia and specifically binds to the hypoxia response element (HRE) to promote transcriptional activation. Reporter vectors based on HRE elements driving luciferase expression have been designed for longitudinal imaging of hypoxia. For example, Viola et al inoculated mice with breast carcinoma cells transfected with an HIF-1α luciferase reporter construct and treated these animals using cyclophosphamide or paclitaxel. They showed that cyclophosphamide significantly inhibited tumor growth and caused an increase in HIF-1α protein levels as quantified using BLI (Viola, Provenzale et al. 2008). As discussed above, a transgenic mouse model was generated in which a chimeric protein consisting of HIF-1α oxygen-dependent degradation domain (ODD) is fused to luciferase. Hypoxic stress lead to the accumulation of ODD-luciferase which could then be identified by non-invasive BL measurement (Goldman, Chen et al. 2011).

Hypoxia stimulates secretion of vascular endothelial growth factor (VEGF) which in turn promotes angiogenesis. Transgenic mice have been engineered to express the VEGF receptor

the DEVD sites were cleaved, luciferase was able to fold appropriately and upon exposure to luciferin, BL photons were produced. Therefore, apoptosis was successfully imaged noninvasively using BLI (Laxman, Hall et al. 2002). Using another methodology, Niers et al engineered the naturally secreted G-Luc so that it is separated by the DEVD sequence. They showed that this fusion protein was retained in the cytoplasm of transfected cells in an inactive form. Upon induction of apoptosis, the DEVD peptide was cleaved in response to caspase-3 activation, freeing G-Luc, which then entered the secretory pathway where it was folded properly and released from the cells. The G-Luc can be detected in the conditioned medium in culture or in blood from live animals (Niers, Kerami et al. 2011). Scabini et al 2011 use a similar approach however in this case a formulated Z-DEVD-aminoluciferin is delivered intraperiotneal to mice carrying human colon cancer or human glioblastoma cell lines engineered to express luciferase. Upon induction of apoptosis Z-DEVD-aminoluciferin is cleaved by caspase 3/7 releasing aminoluciferin that is now free to react with luciferase to generate measurable BL. This group was able to show that after camptothecin and temozolomide treatment of xenograft mouse models of colon cancer and glioblastoma respectively, the treated mice showed higher induction of Z-DEVD-aminoluciferin luminescent signal when compared to the untreated group. Combining D-luciferin that measures the total tumor burden, with Z-DEVD-aminoluciferin that assesses apoptosis induction via caspase activation, they were able to relate inhibition of tumor growth with induction of apoptosis after treatment in the same animal over time (Scabini, Stellari et al. 2011). Hickson et al use the same methodology in a luciferase positive ovarian cancer and breast cancer model. In these experiments, tumor cells were inoculated and allowed to establish, subsequently animals were treated with docetaxel. Animals were injected with the Z-DEVD-aminoluciferin before BL images were acquired. This group shows that more light was detected in the docetaxel-treated group compared with the untreated group (Hickson,

Oxygen is needed for proper cellular metabolism, thus hypoxia, which is common in proliferating cancers, can significantly alter tumor biology on a molecular level. Monitoring hypoxia in vivo can provide important information on tumor biology and response to treatment. The transcription factor Hypoxia-inducing factor 1 (HIF1), is induced under conditions of hypoxia and specifically binds to the hypoxia response element (HRE) to promote transcriptional activation. Reporter vectors based on HRE elements driving luciferase expression have been designed for longitudinal imaging of hypoxia. For example, Viola et al inoculated mice with breast carcinoma cells transfected with an HIF-1α luciferase reporter construct and treated these animals using cyclophosphamide or paclitaxel. They showed that cyclophosphamide significantly inhibited tumor growth and caused an increase in HIF-1α protein levels as quantified using BLI (Viola, Provenzale et al. 2008). As discussed above, a transgenic mouse model was generated in which a chimeric protein consisting of HIF-1α oxygen-dependent degradation domain (ODD) is fused to luciferase. Hypoxic stress lead to the accumulation of ODD-luciferase which could then be identified

Hypoxia stimulates secretion of vascular endothelial growth factor (VEGF) which in turn promotes angiogenesis. Transgenic mice have been engineered to express the VEGF receptor

Ackler et al. 2010).

**5.2.2 Imaging tumor hypoxia and angiogenesis** 

by non-invasive BL measurement (Goldman, Chen et al. 2011).

2 (VEGFR2) promoter that drives F-Luc expression. This mouse model can be used to monitor angiogenesis induced by tumors. Angst et al sought to investigate pancreatic cancer angiogenesis and thus employed the VEGFR2-Luc mouse. After orthotopic inoculation of pancreatic cells, light emission corresponding to VEGFR activity began at day 4, which this group suggests is likely due to wound healing, and continued throughout the experimental period during tumor growth suggesting angiogenesis was occurring. The BL results were confirmed using immunohistochemical staining for CD31 (Angst, Chen et al. 2010). In 2007, Faley et al generated a transgenic reporter mouse, VEGF-GFP/Luc, in which an enhanced green fluorescent protein-luciferase fusion protein is expressed under the control of a human VEGF-A promoter. The VEGF-GFP/Luc animals exhibited intense BL throughout the body at 1 week of age, but the signals declined as the mice grew so that the adult VEGF-GFP/Luc mouse showed BL only in areas undergoing active wound healing. However, in VEGF-GFP/Luc/MMTV mice, BL is observed in spontaneous tumors indicative of active angiogenesis (Faley, Takahashi et al. 2007).

### **5.2.3 Imaging Protein – Protein interactions and cell signalling**

In order to have a mechanistic understanding of tumor biology and response to therapy, oncology research focuses on molecular alterations in the tumor or microenvironment. Under many circumstances up-regulation of oncogenes results in changes in protein–protein interactions, alterations in kinase activity and associated changes in important signalling pathways that promote tumour cell survival and proliferation. Much work has been accomplished to study these signalling cascades in vitro and in ex vivo tissue samples and as a result many therapies have been developed to target these dysregulated pathways. For these reasons there has been a great deal of interest in developing methods to visualize molecular changes in live animals.

Three general methods are currently available for imaging protein-protein interactions in living subjects using reporter genes: a modified mammalian two-hybrid system, a bioluminescence resonance energy transfer (BRET) system, and split reporter protein complementation and reconstitution strategies, these methods were reviewed by Massoud et al in 2007 (Massoud, Paulmurugan et al. 2007). Paulmurgan developed the split reporter system in vivo using very strongly interacting proteins MyoD and Id (Paulmurugan, Umezawa et al. 2002). In 2004 this same group used split synthetic R-Luc protein to evaluate heterodimerization of FRB and FKBP12 mediated by rapamycin. The rapamycin-mediated dimerization of FRB and FKBP12 was studied in living mice by locating, quantifying, and timing the R-Luc BL. Their work demonstrates that the split reporter system can be used to screen small molecule drugs that impact protein-protein interactions in living animals (Paulmurugan, Massoud et al. 2004).

It is also possible to use BLI for the evaluation of enzymatic activity such as kinase activity, in vivo. Khan et al established a luciferase-based reporter to image EGFR kinase activity in an in vivo model of squamous cell carcinoma (SCC). The EGFR Kinase reporter (EKR) is a multidomain chimeric reporter where BL can be used as a marker for EGFR kinase activity. The reporter is phosphorylated in the presence of active EGFR which interferes with luciferase activity, if the substrate is not phosphorylated BL is available for imaging. This reporter can therefore be used as an indicator for EGFR inhibition. Khan et al demonstrated

Bioluminescence Applications in Preclinical Oncology Research 159

treatment regimens, protein-protein interactions, and to gain a better understanding of basic cancer biology. BLI facilitates visualization of processes such as metastasis, angiogenesis, apoptosis and cell signaling in vivo. As noted by Badr et al, the sensitivity of BLI allows for the early detection of tumors and therefore can be useful in the design of preclinical studies assessing prevention strategies (Badr and Tannous 2011). As the BLI modality becomes more popular, work is being done to improve the technology in order to optimize the sensitivity and detection of BL photons. For example, IVIS by Caliper has introduced a system where CT scans and BLI can be used simultaneously to generate three-dimensional images of animals and their disease. Other groups are working on engineering novel luciferases and luciferins to enhance their stability and pharmacokinetics in vivo. As indicated, it is recognized that BLI faces some challenges (distribution and absorption of the substrate as well as scattering issues effecting quantification), however continued use of BLI and proper preclinical study design can overcome most of the problems associated with this modality. BLI as a small animal imaging modality will be an integral part of the future of pre-clinical oncology research and its applications are being refined to achieve an understanding of disease development and response to therapy that was not previously

Ahmann, F. R., H. S. Garewal, et al. (1987). "Intracellular adenosine triphosphate as a

Ahn, B. C., J. A. Ronald, et al. (2011). "Potent, tumor-specific gene expression in an

Angst, E., M. Chen, et al. (2010). "Bioluminescence imaging of angiogenesis in a murine orthotopic pancreatic cancer model." *Mol Imaging Biol* 12(6): 570-575. Badr, C. E., J. M. Niers, et al. (2011). "Suicidal gene therapy in an NF-kappaB-controlled

Badr, C. E. and B. A. Tannous (2011). "Bioluminescence imaging: progress and applications."

Bevis, K. S., L. R. McNally, et al. (2011). "Anti-tumor activity of an anti-DR5 monoclonal

Bhaumik, S. and S. S. Gambhir (2002). "Optical imaging of Renilla luciferase reporter gene

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Briat, A. and G. Vassaux (2008). "A new transgenic mouse line to image chemically induced

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Baert, A. L. (2008). *Encyclopedia of Diagnostic Imaging*, Springer Reference.

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using a novel transgenic mouse model." *Cell Mol Life Sci*.

p53 activation in vivo." *Cancer Sci* 99(4): 683-688.

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orthotopic hepatoma rat model using a Survivin-targeted, amplifiable adenoviral

tumor environment as monitored by a secreted blood reporter." *Gene Ther* 18(5):

antibody, TRA-8, in combination with taxane/platinum-based chemotherapy in an

mapping of metabolites with quantitative bioluminescence." *Radiother Oncol* 99(3):

possible.

**8. References** 

445-451.

398-403.

*Trends Biotechnol*.

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vector." *Gene Ther* 18(6): 606-612.

that a small molecule inhibitor of EGFR kinase activity (erlotinib) was able to inhibit kinase activity in the SSC tumor model using BLI (Khan, Contessa et al. 2011).

BLI has also been used to monitor cell cycle signaling. In vivo BLI can be used to visualize the accumulation of p27-Luc in human tumor cells after the administration of Cdk2 inhibitory drugs (Zhang and Kaelin 2005). Briat et al have generated luciferase-based p53 reporter animals to monitor p53 activation. They showed that in response to doxorubicin induced DNA damage, female animals had weak p53 luciferase activity in the oral cavity while in males, the signal increased in the lower abdominal region (Briat and Vassaux 2008). A reporter molecule has also been developed to measure Akt activity in animals via BLI. The reporter comprises of an engineered luciferase molecule that undergoes a conformational change and gains functionality in response to phosphorylation by Akt (Zhang, Lee et al. 2007).

#### **6. BLI in the study of gene activity, delivery and silencing**

BLI provides a means to study gene delivery, activation using inducible systems, or silencing of tumor promoting genes using RNA interference (RNAi). Delivery of genes can be accomplished using multiple strategies, such as bacterial or viral vector delivery systems, immune cell and stem cell based delivery systems or encapsulation using special nanoparticle formulations such as liposomes or glucosylated polyethyleneimine. Monitoring gene delivery using BLI has also been accomplished. For example Hu et al were able to monitor TGF β receptor gene therapy efficacy in luciferase positive breast cancer metastases simply by monitoring metastases development after gene delivery (Hu, Gerseny et al. 2011). BLI also enables the evaluation of delivery itself. For example, Badr et al have made a construct that comprises of 1) G-Luc, 2) the therapeutic gene cytosine deaminase and 3) uracil phosphoribosyltransferase which converts the nontoxic compound 5-fluorocytosine (5FC) into the drug 5-fluorouracil. A glioma cell line was engineered to express F-Luc. When the constructed gene transfers into tumors, G-Luc allows monitoring of the duration and magnitude of transgene expression while F-Luc imaging was used to monitor tumor growth and response to therapy with the pro-drug 5FC (Badr, Niers et al. 2011). Ahn et al made an adenoviral vector construct where the Survivin promoter (pSurv) amplifies the expression of both the reporter gene F-Luc and therapeutic gene TRAIL. In an orthotopic hepatocellular carcinoma (HCC) rat model, they showed that after systemic administration of the vector, BLI revealed increased F-Luc activity within the tumor compared with the liver indicating that the vector shows tumor-specific transgene expression (Ahn, Ronald et al. 2011). From a gene silencing standpoint, use of luciferase-targeting siRNAs has been studied to define the proof of principle that lipid based systemic administration of luciferase targeting siRNA is able to silence luciferase gene expression in glioma (Ofek, Fischer et al. 2010) and bone metastases (Takeshita, Hokaiwado et al. 2009).

#### **7. Conclusion**

BLI is a well-established tool in cancer research that can provide valuable insight into biological processes in intact cells, excised tissues as well as in animal models of cancer. It can facilitate medium-throughput assessments, it is very sensitive, and reasonably noninvasive. The utility of BLI surpasses simple surveying of tumor growth. More specifically, BLI can be used in the development of sophisticated animal models that examine minimal or metastatic disease, therapeutic efficacy, disease relapse, mechanistic assessments of new treatment regimens, protein-protein interactions, and to gain a better understanding of basic cancer biology. BLI facilitates visualization of processes such as metastasis, angiogenesis, apoptosis and cell signaling in vivo. As noted by Badr et al, the sensitivity of BLI allows for the early detection of tumors and therefore can be useful in the design of preclinical studies assessing prevention strategies (Badr and Tannous 2011). As the BLI modality becomes more popular, work is being done to improve the technology in order to optimize the sensitivity and detection of BL photons. For example, IVIS by Caliper has introduced a system where CT scans and BLI can be used simultaneously to generate three-dimensional images of animals and their disease. Other groups are working on engineering novel luciferases and luciferins to enhance their stability and pharmacokinetics in vivo. As indicated, it is recognized that BLI faces some challenges (distribution and absorption of the substrate as well as scattering issues effecting quantification), however continued use of BLI and proper preclinical study design can overcome most of the problems associated with this modality. BLI as a small animal imaging modality will be an integral part of the future of pre-clinical oncology research and its applications are being refined to achieve an understanding of disease development and response to therapy that was not previously possible.

#### **8. References**

158 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

that a small molecule inhibitor of EGFR kinase activity (erlotinib) was able to inhibit kinase

BLI has also been used to monitor cell cycle signaling. In vivo BLI can be used to visualize the accumulation of p27-Luc in human tumor cells after the administration of Cdk2 inhibitory drugs (Zhang and Kaelin 2005). Briat et al have generated luciferase-based p53 reporter animals to monitor p53 activation. They showed that in response to doxorubicin induced DNA damage, female animals had weak p53 luciferase activity in the oral cavity while in males, the signal increased in the lower abdominal region (Briat and Vassaux 2008). A reporter molecule has also been developed to measure Akt activity in animals via BLI. The reporter comprises of an engineered luciferase molecule that undergoes a conformational change and gains functionality in response to phosphorylation by Akt

BLI provides a means to study gene delivery, activation using inducible systems, or silencing of tumor promoting genes using RNA interference (RNAi). Delivery of genes can be accomplished using multiple strategies, such as bacterial or viral vector delivery systems, immune cell and stem cell based delivery systems or encapsulation using special nanoparticle formulations such as liposomes or glucosylated polyethyleneimine. Monitoring gene delivery using BLI has also been accomplished. For example Hu et al were able to monitor TGF β receptor gene therapy efficacy in luciferase positive breast cancer metastases simply by monitoring metastases development after gene delivery (Hu, Gerseny et al. 2011). BLI also enables the evaluation of delivery itself. For example, Badr et al have made a construct that comprises of 1) G-Luc, 2) the therapeutic gene cytosine deaminase and 3) uracil phosphoribosyltransferase which converts the nontoxic compound 5-fluorocytosine (5FC) into the drug 5-fluorouracil. A glioma cell line was engineered to express F-Luc. When the constructed gene transfers into tumors, G-Luc allows monitoring of the duration and magnitude of transgene expression while F-Luc imaging was used to monitor tumor growth and response to therapy with the pro-drug 5FC (Badr, Niers et al. 2011). Ahn et al made an adenoviral vector construct where the Survivin promoter (pSurv) amplifies the expression of both the reporter gene F-Luc and therapeutic gene TRAIL. In an orthotopic hepatocellular carcinoma (HCC) rat model, they showed that after systemic administration of the vector, BLI revealed increased F-Luc activity within the tumor compared with the liver indicating that the vector shows tumor-specific transgene expression (Ahn, Ronald et al. 2011). From a gene silencing standpoint, use of luciferase-targeting siRNAs has been studied to define the proof of principle that lipid based systemic administration of luciferase targeting siRNA is able to silence luciferase gene expression in glioma (Ofek, Fischer et al. 2010) and bone

BLI is a well-established tool in cancer research that can provide valuable insight into biological processes in intact cells, excised tissues as well as in animal models of cancer. It can facilitate medium-throughput assessments, it is very sensitive, and reasonably noninvasive. The utility of BLI surpasses simple surveying of tumor growth. More specifically, BLI can be used in the development of sophisticated animal models that examine minimal or metastatic disease, therapeutic efficacy, disease relapse, mechanistic assessments of new

activity in the SSC tumor model using BLI (Khan, Contessa et al. 2011).

**6. BLI in the study of gene activity, delivery and silencing** 

metastases (Takeshita, Hokaiwado et al. 2009).

**7. Conclusion** 

(Zhang, Lee et al. 2007).


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**Part 3** 

**Bacterial Bioluminescence** 

