Limits...
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.

Show MeSH

Related in: MedlinePlus

Correlation between BLI, US and direct tumor volume determination in hepatic tumors. MC38Luc1 cells (5 × 105) were injected in the liver of C57BL/6 mice (n = 20), and tumor progression was evaluated weekly by either BLI, US or direct tumor measurement through laparotomy or necropsy. A – Tumor volume determined by direct measurement (calliper, black circles) or US (black squares) over time. Volumes are indicated in the left Y axis. Light emission quantified by BLI is represented as a dotted line and the scale corresponds to (photons/s) × 103 in the right Y axis. Error bars represent standard deviations. B.-Correlation between tumor volumes determined by US (X axis) and direct measurement after necropsy (calliper, Y axis) one month after cell implantation. C-D – Correlation between light emission and direct tumor measurement 2 weeks (C) or 3 weeks (D) after cell implantation (log10 conversion of values).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2648940&req=5

Figure 3: Correlation between BLI, US and direct tumor volume determination in hepatic tumors. MC38Luc1 cells (5 × 105) were injected in the liver of C57BL/6 mice (n = 20), and tumor progression was evaluated weekly by either BLI, US or direct tumor measurement through laparotomy or necropsy. A – Tumor volume determined by direct measurement (calliper, black circles) or US (black squares) over time. Volumes are indicated in the left Y axis. Light emission quantified by BLI is represented as a dotted line and the scale corresponds to (photons/s) × 103 in the right Y axis. Error bars represent standard deviations. B.-Correlation between tumor volumes determined by US (X axis) and direct measurement after necropsy (calliper, Y axis) one month after cell implantation. C-D – Correlation between light emission and direct tumor measurement 2 weeks (C) or 3 weeks (D) after cell implantation (log10 conversion of values).

Mentions: We inoculated 5 × 105 MC38Luc1 cells in the liver of C57BL/6 mice and monitored tumor progression by BLI or US. Subsets of animals in the group were laparotomized or sacrificed at one week intervals for direct calliper measurement of tumor diameters. In figure 2A, we show representative examples of colorectal metastases identified by the three methods at different stages of the disease. Both BLI and US were able to detect the presence and localization of lesions, and to assess their progression over time. US was less sensitive and could not detect the small tumors rising one week after cell injection. However, this technique provided more anatomical information about tumor spread and involvement of other organs. As expected, BLI had lower spatial resolution, but it was able to indicate the approximate location and size of tumors in most of the cases, apart from the light emission quantification. In fact, in those few animals that survived more than 5 weeks, lung metastases usually occurred and could be detected by BLI (figure 2B). Finally, BLI is faster (at least 10 mice can be analyzed at a time in less than 20 minutes) and requires less technical training than US. In order to validate the noninvasive imaging of liver metastases, we analyzed the correlation between BLI, US and direct tumor measurement. In figure 3A we show the progression of the average tumor volume determined by laparotomy or US, as well as the luciferase activity (light emission in photons/s). From this comparison, we can see that tumor progression can be monitored using both BLI and US. In agreement with previous results from other groups [22], BLI is the most sensitive technique, because tumor cells can be detected as early as 2 days after implantation, before they form macroscopic tumors. The maximum background light emission detected in a non-tumor bearing animal was 10,000 photons/s, approximately 10 times less than the average emission of MC38Luc1-injected animals. US started to detect hepatic lesion 2 weeks later. In our experience, tumor diameters determined by US are smaller than those obtained by direct calliper measurement and therefore the estimated volume of tumors is reduced. We believe this is due to the restrictive criteria used to define tumor margins in the US and does not affect the validity of the technique. In fact, correlation between tumor volumes calculated by both methods is good (figure 3B).


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)

Correlation between BLI, US and direct tumor volume determination in hepatic tumors. MC38Luc1 cells (5 × 105) were injected in the liver of C57BL/6 mice (n = 20), and tumor progression was evaluated weekly by either BLI, US or direct tumor measurement through laparotomy or necropsy. A – Tumor volume determined by direct measurement (calliper, black circles) or US (black squares) over time. Volumes are indicated in the left Y axis. Light emission quantified by BLI is represented as a dotted line and the scale corresponds to (photons/s) × 103 in the right Y axis. Error bars represent standard deviations. B.-Correlation between tumor volumes determined by US (X axis) and direct measurement after necropsy (calliper, Y axis) one month after cell implantation. C-D – Correlation between light emission and direct tumor measurement 2 weeks (C) or 3 weeks (D) after cell implantation (log10 conversion of values).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Correlation between BLI, US and direct tumor volume determination in hepatic tumors. MC38Luc1 cells (5 × 105) were injected in the liver of C57BL/6 mice (n = 20), and tumor progression was evaluated weekly by either BLI, US or direct tumor measurement through laparotomy or necropsy. A – Tumor volume determined by direct measurement (calliper, black circles) or US (black squares) over time. Volumes are indicated in the left Y axis. Light emission quantified by BLI is represented as a dotted line and the scale corresponds to (photons/s) × 103 in the right Y axis. Error bars represent standard deviations. B.-Correlation between tumor volumes determined by US (X axis) and direct measurement after necropsy (calliper, Y axis) one month after cell implantation. C-D – Correlation between light emission and direct tumor measurement 2 weeks (C) or 3 weeks (D) after cell implantation (log10 conversion of values).
Mentions: We inoculated 5 × 105 MC38Luc1 cells in the liver of C57BL/6 mice and monitored tumor progression by BLI or US. Subsets of animals in the group were laparotomized or sacrificed at one week intervals for direct calliper measurement of tumor diameters. In figure 2A, we show representative examples of colorectal metastases identified by the three methods at different stages of the disease. Both BLI and US were able to detect the presence and localization of lesions, and to assess their progression over time. US was less sensitive and could not detect the small tumors rising one week after cell injection. However, this technique provided more anatomical information about tumor spread and involvement of other organs. As expected, BLI had lower spatial resolution, but it was able to indicate the approximate location and size of tumors in most of the cases, apart from the light emission quantification. In fact, in those few animals that survived more than 5 weeks, lung metastases usually occurred and could be detected by BLI (figure 2B). Finally, BLI is faster (at least 10 mice can be analyzed at a time in less than 20 minutes) and requires less technical training than US. In order to validate the noninvasive imaging of liver metastases, we analyzed the correlation between BLI, US and direct tumor measurement. In figure 3A we show the progression of the average tumor volume determined by laparotomy or US, as well as the luciferase activity (light emission in photons/s). From this comparison, we can see that tumor progression can be monitored using both BLI and US. In agreement with previous results from other groups [22], BLI is the most sensitive technique, because tumor cells can be detected as early as 2 days after implantation, before they form macroscopic tumors. The maximum background light emission detected in a non-tumor bearing animal was 10,000 photons/s, approximately 10 times less than the average emission of MC38Luc1-injected animals. US started to detect hepatic lesion 2 weeks later. In our experience, tumor diameters determined by US are smaller than those obtained by direct calliper measurement and therefore the estimated volume of tumors is reduced. We believe this is due to the restrictive criteria used to define tumor margins in the US and does not affect the validity of the technique. In fact, correlation between tumor volumes calculated by both methods is good (figure 3B).

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