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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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Analysis of luciferase stability and function in vivo. A. – Intrahepatic MC38Luc1 tumors (n = 10) were excised at different times after cell implantation (2, 3 and 4 weeks), and the relative copy number of the plasmid pCDNA3-luc was determined by qPCR. The values correspond to the number of plasmid copies divided by the copies of the endogenous albumin gene. Error bars represent standard deviations. B. – Correlation between the relative pCDNA3-luc copy number in advanced tumors (4 weeks after implantation) and tumor volume. C. – Correlation between in vivo light emission of advanced tumors (in photons/sec.) and luciferase activity (in RLUs) obtained in solution from tumor extracts (log10 conversion of values).
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Figure 5: Analysis of luciferase stability and function in vivo. A. – Intrahepatic MC38Luc1 tumors (n = 10) were excised at different times after cell implantation (2, 3 and 4 weeks), and the relative copy number of the plasmid pCDNA3-luc was determined by qPCR. The values correspond to the number of plasmid copies divided by the copies of the endogenous albumin gene. Error bars represent standard deviations. B. – Correlation between the relative pCDNA3-luc copy number in advanced tumors (4 weeks after implantation) and tumor volume. C. – Correlation between in vivo light emission of advanced tumors (in photons/sec.) and luciferase activity (in RLUs) obtained in solution from tumor extracts (log10 conversion of values).

Mentions: It is not clear at this moment if the limitations of BLI in large tumors can be considered a general characteristic of this technique. The observation periods described in the literature are heterogeneous, and the time to reach the "advanced stage" differs in each tumor model. Nevertheless, attenuation of luciferase signal has been previously described in another colorectal cancer liver metastasis model developed in Balb/c mice [27]. A partial loss of correlation between BLI and physical measurement was found in large tumors starting on day 20 after cell inoculation, which is similar to our observations. In this case the main reason was the quenching effect of the ascitic fluid, which is usually abundant in advanced CT26 tumors growing in the liver. Accumulation of large volumes of hemorrhagic ascites was less frequent in our intrahepatic MC38Luc1 tumors. In addition, this factor cannot play a role in the subcutaneous tumors described here. In a different study, a marked inhibition of bioluminescence was found in a bladder cancer model, starting 2 weeks after cell implantation [34]. The authors demonstrated a reduction in light emission from necrotic and hemorrhagic areas of the tumors. In addition, these areas can contribute to the quenching effect of surrounding tissues. If the proportion of these inactive fractions of the tumor increases over time and is more prevalent in large tumors, this is a feasible explanation for the loss of correlation between bioluminescence and tumor volume. In our model, histological analysis of tumors revealed variable regions of necrosis and haemorrhage. However, when we studied individual tumors, we could not establish a direct correlation between the abundance of these areas and the decline of light emission (data not shown). Therefore, other factors may play a role in this phenomenon. We have verified by quantitative PCR that there is no decrease in the frequency of luciferase plasmids integrated in the tumors at different time points. As shown in figure 5A, we found an average of approximately 4 copies of plasmid for every copy of the endogenous albumin gene (which means 2 copies per genome) even in the very advanced stage (day 28). This indicates that the MC38Luc1 cells do not lose the luciferase gene over time in vivo, and the proportion of these cells in the tumor mass is maintained on average. Interestingly, the ratio of copies/genome is not randomly distributed among tumors at day 28 (figure 5B). We observed that larger tumors tend to present less copies of luciferase plasmid per genome, suggesting that stromal cells (fibroblasts, endothelial cells, etc.), which do not contribute to the luciferase expression are more abundant in these tumors. These luciferase-inactive components of the tumors, together with areas of necrosis and haemorrhage are mixed in variable proportions and impair an accurate determination of tumor volume by BLI. Therefore, in advanced stages of our tumor model, the functional information obtained by BLI prevails over the morphological criteria.


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)

Analysis of luciferase stability and function in vivo. A. – Intrahepatic MC38Luc1 tumors (n = 10) were excised at different times after cell implantation (2, 3 and 4 weeks), and the relative copy number of the plasmid pCDNA3-luc was determined by qPCR. The values correspond to the number of plasmid copies divided by the copies of the endogenous albumin gene. Error bars represent standard deviations. B. – Correlation between the relative pCDNA3-luc copy number in advanced tumors (4 weeks after implantation) and tumor volume. C. – Correlation between in vivo light emission of advanced tumors (in photons/sec.) and luciferase activity (in RLUs) obtained in solution from tumor extracts (log10 conversion of values).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2648940&req=5

Figure 5: Analysis of luciferase stability and function in vivo. A. – Intrahepatic MC38Luc1 tumors (n = 10) were excised at different times after cell implantation (2, 3 and 4 weeks), and the relative copy number of the plasmid pCDNA3-luc was determined by qPCR. The values correspond to the number of plasmid copies divided by the copies of the endogenous albumin gene. Error bars represent standard deviations. B. – Correlation between the relative pCDNA3-luc copy number in advanced tumors (4 weeks after implantation) and tumor volume. C. – Correlation between in vivo light emission of advanced tumors (in photons/sec.) and luciferase activity (in RLUs) obtained in solution from tumor extracts (log10 conversion of values).
Mentions: It is not clear at this moment if the limitations of BLI in large tumors can be considered a general characteristic of this technique. The observation periods described in the literature are heterogeneous, and the time to reach the "advanced stage" differs in each tumor model. Nevertheless, attenuation of luciferase signal has been previously described in another colorectal cancer liver metastasis model developed in Balb/c mice [27]. A partial loss of correlation between BLI and physical measurement was found in large tumors starting on day 20 after cell inoculation, which is similar to our observations. In this case the main reason was the quenching effect of the ascitic fluid, which is usually abundant in advanced CT26 tumors growing in the liver. Accumulation of large volumes of hemorrhagic ascites was less frequent in our intrahepatic MC38Luc1 tumors. In addition, this factor cannot play a role in the subcutaneous tumors described here. In a different study, a marked inhibition of bioluminescence was found in a bladder cancer model, starting 2 weeks after cell implantation [34]. The authors demonstrated a reduction in light emission from necrotic and hemorrhagic areas of the tumors. In addition, these areas can contribute to the quenching effect of surrounding tissues. If the proportion of these inactive fractions of the tumor increases over time and is more prevalent in large tumors, this is a feasible explanation for the loss of correlation between bioluminescence and tumor volume. In our model, histological analysis of tumors revealed variable regions of necrosis and haemorrhage. However, when we studied individual tumors, we could not establish a direct correlation between the abundance of these areas and the decline of light emission (data not shown). Therefore, other factors may play a role in this phenomenon. We have verified by quantitative PCR that there is no decrease in the frequency of luciferase plasmids integrated in the tumors at different time points. As shown in figure 5A, we found an average of approximately 4 copies of plasmid for every copy of the endogenous albumin gene (which means 2 copies per genome) even in the very advanced stage (day 28). This indicates that the MC38Luc1 cells do not lose the luciferase gene over time in vivo, and the proportion of these cells in the tumor mass is maintained on average. Interestingly, the ratio of copies/genome is not randomly distributed among tumors at day 28 (figure 5B). We observed that larger tumors tend to present less copies of luciferase plasmid per genome, suggesting that stromal cells (fibroblasts, endothelial cells, etc.), which do not contribute to the luciferase expression are more abundant in these tumors. These luciferase-inactive components of the tumors, together with areas of necrosis and haemorrhage are mixed in variable proportions and impair an accurate determination of tumor volume by BLI. Therefore, in advanced stages of our tumor model, the functional information obtained by BLI prevails over the morphological criteria.

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