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Evaluation of bioluminescent imaging for noninvasive monitoring of colorectal cancer progression in the liver and its response to immunogene therapy.

Zabala M, Alzuguren P, Benavides C, Crettaz J, Gonzalez-Aseguinolaza G, Ortiz de Solorzano C, Gonzalez-Aparicio M, Kramer MG, Prieto J, Hernandez-Alcoceba R - Mol. Cancer (2009)

Bottom Line: Individualized quantification of light emission was able to determine the extent and duration of antitumor responses and to predict long-term disease-free survival.We show that BLI is a rapid, convenient and safe technique for the individual monitorization of tumor progression in the liver.Evaluation of experimental treatments with complex mechanisms of action such as immunotherapy is possible using this technology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Gene Therapy and Hepatology, CIMA, University of Navarra, Foundation for Applied Medical Research, Pamplona, Spain. mzabala@stanford.edu

ABSTRACT

Background: Bioluminescent imaging (BLI) is based on the detection of light emitted by living cells expressing a luciferase gene. Stable transfection of luciferase in cancer cells and their inoculation into permissive animals allows the noninvasive monitorization of tumor progression inside internal organs. We have applied this technology for the development of a murine model of colorectal cancer involving the liver, with the aim of improving the pre-clinical evaluation of new anticancer therapies.

Results: A murine colon cancer cell line stably transfected with the luciferase gene (MC38Luc1) retains tumorigenicity in immunocompetent C57BL/6 animals. Intrahepatic inoculation of MC38Luc1 causes progressive liver infiltration that can be monitored by BLI. Compared with ultrasonography (US), BLI is more sensitive, but accurate estimation of tumor mass is impaired in advanced stages. We applied BLI to evaluate the efficacy of an immunogene therapy approach based on the liver-specific expression of the proinflammatory cytokine interleukin-12 (IL-12). Individualized quantification of light emission was able to determine the extent and duration of antitumor responses and to predict long-term disease-free survival.

Conclusion: We show that BLI is a rapid, convenient and safe technique for the individual monitorization of tumor progression in the liver. Evaluation of experimental treatments with complex mechanisms of action such as immunotherapy is possible using this technology.

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Antitumor effect of IL-12 monitored by BLI. MC38Luc1 cells (5 × 105) were injected in the liver of C57BL/6 mice (n = 26). After verification of cell engraftment, the vector GL-Ad/RUmIL-12 was injected intravenously at 2.5 × 108 iu/mouse in half of the mice, and IL-12 expression was activated by mifepristone one week later. A. – Quantification of light emission before virus injection (day 2) and 7 days after initiation of IL-12 expression (day 17) in control (Co) and treated groups (IL-12). B. – Monitoring of tumor progression by BLI. Control group is represented as a dotted line. Three subgroups were defined among treated animals: non-responders (NR), mice with partial response (PR) and complete response (CR). Differences were statistically significant (p < 0.01) between Co and PR groups during days 20 to 30. Light emission from the CR group remained significantly lower until the end of the experiment. C. – Average tumor volume of the different groups one month after cell implantation, and at the time of sacrifice or spontaneous death. No evidence of tumor was observed in the CR group. Error bars represent standard deviations. * p < 0.05; ** p < 0.01.
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Figure 6: Antitumor effect of IL-12 monitored by BLI. MC38Luc1 cells (5 × 105) were injected in the liver of C57BL/6 mice (n = 26). After verification of cell engraftment, the vector GL-Ad/RUmIL-12 was injected intravenously at 2.5 × 108 iu/mouse in half of the mice, and IL-12 expression was activated by mifepristone one week later. A. – Quantification of light emission before virus injection (day 2) and 7 days after initiation of IL-12 expression (day 17) in control (Co) and treated groups (IL-12). B. – Monitoring of tumor progression by BLI. Control group is represented as a dotted line. Three subgroups were defined among treated animals: non-responders (NR), mice with partial response (PR) and complete response (CR). Differences were statistically significant (p < 0.01) between Co and PR groups during days 20 to 30. Light emission from the CR group remained significantly lower until the end of the experiment. C. – Average tumor volume of the different groups one month after cell implantation, and at the time of sacrifice or spontaneous death. No evidence of tumor was observed in the CR group. Error bars represent standard deviations. * p < 0.05; ** p < 0.01.

Mentions: The ability of BLI to evaluate the efficacy of conventional [24] or experimental [30,31] treatments has been demonstrated in different tumor models, but little is known about immunotherapy approaches in syngeneic mice. We have previously described a potent antitumor effect of the high-capacity adenoviral vector GL-Ad/RUmIL-12 on intrahepatic MC38-derived tumors [11]. A liver-specific, Mifepristone-inducible expression system allows controlled expression of IL-12. The vector was administered one week before cell implantation, and activation of IL-12 expression started 5 days later. Under these circumstances, tumor eradication was observed in most of the animals when high doses of IL-12 were achieved. Now we aim to evaluate the efficacy of GL-Ad/RUmIL-12 in a more restrictive setting. BLI was used with the objective of determining the onset and duration of the antitumor response in each animal. In the actual protocol, tumor cells (MC38Luc1) were implanted before administration of the virus. Initial BLI performed 48 hours after cell inoculation allowed us to verify the localization of cells in the liver area and the homogeneity of experimental groups in terms of light emission (figure 6A). The next day, a moderate dose of GL-Ad/RUmIL-12 vector (2.5 × 108 iu) was administered intravenously, and induction of IL-12 expression started one week later. Therefore, tumors had progressed for 10 days before the treatment effectively started. The induction regime consisted on 10 daily injections of 250 μg/kg Mifepristone intraperitoneally, as previously described [11]. The average concentration of IL-12 in serum was 27 ng/ml after the first mifepristone administration, which is consistent with the dose of virus used. Bioluminescence quantification detected significant differences between the control and treated groups as early as 7 days after initiation of IL-12 expression (day 17 after cell inoculation, figure 6A). More importantly, subsequent BLI monitorization (figure 6B) allowed us to distinguish 3 subsets of animals inside the treatment group: non-responders (NR), in which light emission was similar to the control group; mice showing a partial response (PR) consisting in a transient inhibition of luciferase activity; and finally others in which the signal was completely and permanently abolished (CR). All animals that survived long enough were laparotomized 4 weeks after initiation of the experiment in order to determine the size of liver tumors by direct calliper measurement. As shown in figure 6C, stratification of mice according to bioluminescence was in accordance with the changes observed in tumor volume. In the PR group, a significant reduction was observed during the 4th week of the experiment, but the tumors progressed and all animals finally died during the next 4 weeks because of their hepatic lesions. In contrast, mice in the CR group remained tumor-free and were negative for luciferase activity for the entire duration of the experiment (more than 6 months).


Evaluation of bioluminescent imaging for noninvasive monitoring of colorectal cancer progression in the liver and its response to immunogene therapy.

Zabala M, Alzuguren P, Benavides C, Crettaz J, Gonzalez-Aseguinolaza G, Ortiz de Solorzano C, Gonzalez-Aparicio M, Kramer MG, Prieto J, Hernandez-Alcoceba R - Mol. Cancer (2009)

Antitumor effect of IL-12 monitored by BLI. MC38Luc1 cells (5 × 105) were injected in the liver of C57BL/6 mice (n = 26). After verification of cell engraftment, the vector GL-Ad/RUmIL-12 was injected intravenously at 2.5 × 108 iu/mouse in half of the mice, and IL-12 expression was activated by mifepristone one week later. A. – Quantification of light emission before virus injection (day 2) and 7 days after initiation of IL-12 expression (day 17) in control (Co) and treated groups (IL-12). B. – Monitoring of tumor progression by BLI. Control group is represented as a dotted line. Three subgroups were defined among treated animals: non-responders (NR), mice with partial response (PR) and complete response (CR). Differences were statistically significant (p < 0.01) between Co and PR groups during days 20 to 30. Light emission from the CR group remained significantly lower until the end of the experiment. C. – Average tumor volume of the different groups one month after cell implantation, and at the time of sacrifice or spontaneous death. No evidence of tumor was observed in the CR group. Error bars represent standard deviations. * p < 0.05; ** p < 0.01.
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Figure 6: Antitumor effect of IL-12 monitored by BLI. MC38Luc1 cells (5 × 105) were injected in the liver of C57BL/6 mice (n = 26). After verification of cell engraftment, the vector GL-Ad/RUmIL-12 was injected intravenously at 2.5 × 108 iu/mouse in half of the mice, and IL-12 expression was activated by mifepristone one week later. A. – Quantification of light emission before virus injection (day 2) and 7 days after initiation of IL-12 expression (day 17) in control (Co) and treated groups (IL-12). B. – Monitoring of tumor progression by BLI. Control group is represented as a dotted line. Three subgroups were defined among treated animals: non-responders (NR), mice with partial response (PR) and complete response (CR). Differences were statistically significant (p < 0.01) between Co and PR groups during days 20 to 30. Light emission from the CR group remained significantly lower until the end of the experiment. C. – Average tumor volume of the different groups one month after cell implantation, and at the time of sacrifice or spontaneous death. No evidence of tumor was observed in the CR group. Error bars represent standard deviations. * p < 0.05; ** p < 0.01.
Mentions: The ability of BLI to evaluate the efficacy of conventional [24] or experimental [30,31] treatments has been demonstrated in different tumor models, but little is known about immunotherapy approaches in syngeneic mice. We have previously described a potent antitumor effect of the high-capacity adenoviral vector GL-Ad/RUmIL-12 on intrahepatic MC38-derived tumors [11]. A liver-specific, Mifepristone-inducible expression system allows controlled expression of IL-12. The vector was administered one week before cell implantation, and activation of IL-12 expression started 5 days later. Under these circumstances, tumor eradication was observed in most of the animals when high doses of IL-12 were achieved. Now we aim to evaluate the efficacy of GL-Ad/RUmIL-12 in a more restrictive setting. BLI was used with the objective of determining the onset and duration of the antitumor response in each animal. In the actual protocol, tumor cells (MC38Luc1) were implanted before administration of the virus. Initial BLI performed 48 hours after cell inoculation allowed us to verify the localization of cells in the liver area and the homogeneity of experimental groups in terms of light emission (figure 6A). The next day, a moderate dose of GL-Ad/RUmIL-12 vector (2.5 × 108 iu) was administered intravenously, and induction of IL-12 expression started one week later. Therefore, tumors had progressed for 10 days before the treatment effectively started. The induction regime consisted on 10 daily injections of 250 μg/kg Mifepristone intraperitoneally, as previously described [11]. The average concentration of IL-12 in serum was 27 ng/ml after the first mifepristone administration, which is consistent with the dose of virus used. Bioluminescence quantification detected significant differences between the control and treated groups as early as 7 days after initiation of IL-12 expression (day 17 after cell inoculation, figure 6A). More importantly, subsequent BLI monitorization (figure 6B) allowed us to distinguish 3 subsets of animals inside the treatment group: non-responders (NR), in which light emission was similar to the control group; mice showing a partial response (PR) consisting in a transient inhibition of luciferase activity; and finally others in which the signal was completely and permanently abolished (CR). All animals that survived long enough were laparotomized 4 weeks after initiation of the experiment in order to determine the size of liver tumors by direct calliper measurement. As shown in figure 6C, stratification of mice according to bioluminescence was in accordance with the changes observed in tumor volume. In the PR group, a significant reduction was observed during the 4th week of the experiment, but the tumors progressed and all animals finally died during the next 4 weeks because of their hepatic lesions. In contrast, mice in the CR group remained tumor-free and were negative for luciferase activity for the entire duration of the experiment (more than 6 months).

Bottom Line: Individualized quantification of light emission was able to determine the extent and duration of antitumor responses and to predict long-term disease-free survival.We show that BLI is a rapid, convenient and safe technique for the individual monitorization of tumor progression in the liver.Evaluation of experimental treatments with complex mechanisms of action such as immunotherapy is possible using this technology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Gene Therapy and Hepatology, CIMA, University of Navarra, Foundation for Applied Medical Research, Pamplona, Spain. mzabala@stanford.edu

ABSTRACT

Background: Bioluminescent imaging (BLI) is based on the detection of light emitted by living cells expressing a luciferase gene. Stable transfection of luciferase in cancer cells and their inoculation into permissive animals allows the noninvasive monitorization of tumor progression inside internal organs. We have applied this technology for the development of a murine model of colorectal cancer involving the liver, with the aim of improving the pre-clinical evaluation of new anticancer therapies.

Results: A murine colon cancer cell line stably transfected with the luciferase gene (MC38Luc1) retains tumorigenicity in immunocompetent C57BL/6 animals. Intrahepatic inoculation of MC38Luc1 causes progressive liver infiltration that can be monitored by BLI. Compared with ultrasonography (US), BLI is more sensitive, but accurate estimation of tumor mass is impaired in advanced stages. We applied BLI to evaluate the efficacy of an immunogene therapy approach based on the liver-specific expression of the proinflammatory cytokine interleukin-12 (IL-12). Individualized quantification of light emission was able to determine the extent and duration of antitumor responses and to predict long-term disease-free survival.

Conclusion: We show that BLI is a rapid, convenient and safe technique for the individual monitorization of tumor progression in the liver. Evaluation of experimental treatments with complex mechanisms of action such as immunotherapy is possible using this technology.

Show MeSH
Related in: MedlinePlus