Non-invasive imaging to reduce and refine the use of animals and monitor their welfare during the course of experimentations in oncology
Lead Research Organisation:
Cancer Research UK
Department Name: London Research Institute (LIF)
Abstract
Despite the efforts of the international community, including ours, faithfully and reliably mimicking human cancers in experimental animal models remains a fundamental requirement to improve our understanding of the disease and develop / assess more effective therapeutic strategies. It is generally admitted that orthotopic models, when cancer cells are located in the organ they originate from, are more informative and reliable than ectopic models (when the cancer cells are forced to grow in place they would normally not use, like under the skin). However, it is very challenging to analyse the tumour progression in orthotopic models since they cannot be seen with naked eyes. As a consequence, a batch of animals needs to be sacrificed at each time point for analysing the organs after dissection (bone marrow and lungs in the case of this project). In vivo imaging technologies offer exciting possibilities to conduct noninvasive and longitudinal studies over time to understand the dynamics of biological processes in live animals over long period of time. The basic idea is to use imaging approaches to quantify the amount of tumours and their distribution within the body of the animal over time, in a way that mimics diagnosis for human patients. In that case there is no need to sacrifice the subject to obtain these measurements and it is then possible to assess the evolution of cancer cells over time. Therefore in vivo imaging allows to reduce the number of animals needed to answer a biological question and / or the efficacy of a new treatment. Furthermore, different imaging techniques can be combined on the same animal in order to answer different questions at the same time instead of doing different experiments.
Xenotransplantation of human acute myeloid leukaemia (AML) initiating cells (AML-ICs) from samples coming directly from patients is a very powerful model to understand the development of human leukaemia and assess response to treatments. However to collect results over time, it is necessary either to sacrifice some animals, or to perform bone marrow biopsies, a well tolerated but sill invasive procedure. We can use bioluminescence (BLI) to track genetically engineered cell lines in the bone marrow, but this cannot be used for patients' samples. To our knowledge, no noninvasive approach has been reported so far to monitor primary human AML over time. We propose here to tests different strategies to achieve this goal, which would allow to decrease the number of animals needed to complete the studies in that field. Furthermore, being able to objectively quantify the "tumour load" would allow one to assess at the same time the health status of the animal, similarly to what is done for patients. Consequently, researchers would have the capability to refine their experimental conditions and stop the experiment as soon as they have obtained the answers they need.
We also use genetically engineered mouse models where a specific gene has been mutated and will induce the spontaneous apparition of a lung cancer, closely mimicking a specific type of human cancer. We can use x-ray computed tomography (CT) in mice to detect lung tumours, like it is done in humans, but we are trying to develop more sensitive techniques and also to collect more information that just the size of the nodules.
Therefore we need to test different types of probes (fluorescently labelled or coupled with radioactive tracers), which could be used to detect by modern in vivo imaging technologies (Near-infrared fluorescence imaging and Cerenkov luminescence imaging) the presence and the progression of the leukaemia / lung tumours. We also want to measure physiologic parameters (heart rate, breathing rate, oxygenation) to assess objectively the welfare of the animals during the course of the study.
Altogether, these different information would allow us to reduce the need for animals and refine the way they are used during the experiments
Xenotransplantation of human acute myeloid leukaemia (AML) initiating cells (AML-ICs) from samples coming directly from patients is a very powerful model to understand the development of human leukaemia and assess response to treatments. However to collect results over time, it is necessary either to sacrifice some animals, or to perform bone marrow biopsies, a well tolerated but sill invasive procedure. We can use bioluminescence (BLI) to track genetically engineered cell lines in the bone marrow, but this cannot be used for patients' samples. To our knowledge, no noninvasive approach has been reported so far to monitor primary human AML over time. We propose here to tests different strategies to achieve this goal, which would allow to decrease the number of animals needed to complete the studies in that field. Furthermore, being able to objectively quantify the "tumour load" would allow one to assess at the same time the health status of the animal, similarly to what is done for patients. Consequently, researchers would have the capability to refine their experimental conditions and stop the experiment as soon as they have obtained the answers they need.
We also use genetically engineered mouse models where a specific gene has been mutated and will induce the spontaneous apparition of a lung cancer, closely mimicking a specific type of human cancer. We can use x-ray computed tomography (CT) in mice to detect lung tumours, like it is done in humans, but we are trying to develop more sensitive techniques and also to collect more information that just the size of the nodules.
Therefore we need to test different types of probes (fluorescently labelled or coupled with radioactive tracers), which could be used to detect by modern in vivo imaging technologies (Near-infrared fluorescence imaging and Cerenkov luminescence imaging) the presence and the progression of the leukaemia / lung tumours. We also want to measure physiologic parameters (heart rate, breathing rate, oxygenation) to assess objectively the welfare of the animals during the course of the study.
Altogether, these different information would allow us to reduce the need for animals and refine the way they are used during the experiments
Technical Summary
We have identified 7 probes for NEAR-INFRARED IMAGING (NIRI) (we are producing 1 of them)
- 2-Deoxy-Glucose (2 sources)
- MMPs sensitive probe
- Cathepsins sensitive probe
- Integrin alphov beta3 probe
- Blood pool agent
- Antibody directed against hCD33 (marker of AML)
Very recent publications have reported that probes labelled with the radionuclide [18F], routinely exploited for positron emission tomography (PET) imaging, can be detected thanks to a technique called CERENKOV LUMINESCENCE IMAGING (CLI). We will evaluate the following PET probes:
- 2-deoxy-2-[18F]fluoro-D-glucose
- [18F]-3-fluorodeoxythymidine
- [18] Fluorine
We will be using a MouseOx system to measure various PHYSIOLOGIC PARAMETERS like peripheral blood oxygen saturation, heart frequency or breathing frequency with a noninvasive sensor clip.
In the earliest stage of the project, we will use 1 or 2 luciferase-expressing leukaemic cell lines so that BLI can be used as a positive control and healthy mice will be used as negative controls. For each probe, 2-3 leukaemia bearing mice and 2 negative controls will receive the probe of interest and be imaged at different time points after administration of the probe. BLI will be co acquired with the NIRI / CLI signal. The operation will be repeated weekly until the apparition of clinical signs.
The general status of the animals will be checked weekly using the MouseOx system. This information, together with BLI, NIRI and CLI quantification, will reinforce our daily observation for clinical signs.
To be selected, a probe will have to show in all leukaemia or tumour bearing mice at least a two-fold increase of the signal as compared to the negative controls. Successful candidates will then be tested on primary human AML and sporadic mouse lung tumours. Eventually we will optimise the procedures in order to allow simultaneous analysis of the successful probes on the same animal to maximise the reduction of animal use.
- 2-Deoxy-Glucose (2 sources)
- MMPs sensitive probe
- Cathepsins sensitive probe
- Integrin alphov beta3 probe
- Blood pool agent
- Antibody directed against hCD33 (marker of AML)
Very recent publications have reported that probes labelled with the radionuclide [18F], routinely exploited for positron emission tomography (PET) imaging, can be detected thanks to a technique called CERENKOV LUMINESCENCE IMAGING (CLI). We will evaluate the following PET probes:
- 2-deoxy-2-[18F]fluoro-D-glucose
- [18F]-3-fluorodeoxythymidine
- [18] Fluorine
We will be using a MouseOx system to measure various PHYSIOLOGIC PARAMETERS like peripheral blood oxygen saturation, heart frequency or breathing frequency with a noninvasive sensor clip.
In the earliest stage of the project, we will use 1 or 2 luciferase-expressing leukaemic cell lines so that BLI can be used as a positive control and healthy mice will be used as negative controls. For each probe, 2-3 leukaemia bearing mice and 2 negative controls will receive the probe of interest and be imaged at different time points after administration of the probe. BLI will be co acquired with the NIRI / CLI signal. The operation will be repeated weekly until the apparition of clinical signs.
The general status of the animals will be checked weekly using the MouseOx system. This information, together with BLI, NIRI and CLI quantification, will reinforce our daily observation for clinical signs.
To be selected, a probe will have to show in all leukaemia or tumour bearing mice at least a two-fold increase of the signal as compared to the negative controls. Successful candidates will then be tested on primary human AML and sporadic mouse lung tumours. Eventually we will optimise the procedures in order to allow simultaneous analysis of the successful probes on the same animal to maximise the reduction of animal use.
Planned Impact
1-Reduction of the number of animals to be used
-Thanks to non-invasive monitoring: In vivo imaging allows performing longitudinal monitoring in a non-invasive way. As compared to an end-point strategy, this leads to a massive reduction of the number of animals to be used to collect the same amount of information. In the case one would need groups of 5 animals per group, 3 groups of treatment and 5 time points of analysis, the endpoint strategy would require 75 mice to be used. The non-invasive longitudinal monitoring will require only 15 mice, each one being analysed 5 times during the course of the study. In this particular case this is an 80% reduction that is achieved thanks to imaging.
-Thanks to "multiplexing" different biomarkers: In many instance it is possible to combine the analysis of different biomarkers within the same animal, and this is one of the ultimate goal of the study. In this case it becomes possible to answer different questions at the same time, on the same animal. For example, if one wants to analyse the consumption of glucose by tumour cells and neo-angiogenesis associated with tumour growth, it would normally take two separate sets of experiments to obtain these information. If we transpose this requirement to the previous scenario (5 animals / group x 3 groups x 5 time points), the endpoint strategy will require 150 mice, while the "multiplexed" non-invasive imaging still needs only 15 mice, i.e. a 90% reduction. Based on the capabilities of our imaging system, we can acquire concomitantly BLI, CLI and 2 NIR probes within the same animal. In parallel, we will also be able to analyse physiologic parameters (oxygen saturation, heart rate and breathing rate).
2-Refinement of the use of animals used in oncology studies
-Objective and quantitative monitoring of animal welfare: in order to minimise discomfort, distress and suffering, it is critically important to be able to quantify these parameters so that appropriate actions can be implemented accordingly. This project basically deals with the development of diagnosis procedures to objectively quantify disease progression and potential consequences on physiologic parameters. The whole idea is to be able to use these diagnosis tools to improve the quality of the data produced and to take advantage of this information to refine the criteria we can use to terminate an experiment. In this context, we hope that some of these biomarkers will be informative of mouse "clinical evolution" before the apparition of visible clinical signs. In this case the experiment could be terminated before the apparition of clinical sign for the mouse.
-Reduce the need for BM aspirates: As previously mentioned, the only way to conduct longitudinal studies with the primary AML model would be to perform sequential bone marrow aspiration from the femur or tibia using a needle. This is an effective but yet invasive approach. We propose here to assess the possibility to collect information from the bone marrow thanks to non-invasive in vivo imaging, which would represent a significant refinement of the experimental procedures.
-Thanks to non-invasive monitoring: In vivo imaging allows performing longitudinal monitoring in a non-invasive way. As compared to an end-point strategy, this leads to a massive reduction of the number of animals to be used to collect the same amount of information. In the case one would need groups of 5 animals per group, 3 groups of treatment and 5 time points of analysis, the endpoint strategy would require 75 mice to be used. The non-invasive longitudinal monitoring will require only 15 mice, each one being analysed 5 times during the course of the study. In this particular case this is an 80% reduction that is achieved thanks to imaging.
-Thanks to "multiplexing" different biomarkers: In many instance it is possible to combine the analysis of different biomarkers within the same animal, and this is one of the ultimate goal of the study. In this case it becomes possible to answer different questions at the same time, on the same animal. For example, if one wants to analyse the consumption of glucose by tumour cells and neo-angiogenesis associated with tumour growth, it would normally take two separate sets of experiments to obtain these information. If we transpose this requirement to the previous scenario (5 animals / group x 3 groups x 5 time points), the endpoint strategy will require 150 mice, while the "multiplexed" non-invasive imaging still needs only 15 mice, i.e. a 90% reduction. Based on the capabilities of our imaging system, we can acquire concomitantly BLI, CLI and 2 NIR probes within the same animal. In parallel, we will also be able to analyse physiologic parameters (oxygen saturation, heart rate and breathing rate).
2-Refinement of the use of animals used in oncology studies
-Objective and quantitative monitoring of animal welfare: in order to minimise discomfort, distress and suffering, it is critically important to be able to quantify these parameters so that appropriate actions can be implemented accordingly. This project basically deals with the development of diagnosis procedures to objectively quantify disease progression and potential consequences on physiologic parameters. The whole idea is to be able to use these diagnosis tools to improve the quality of the data produced and to take advantage of this information to refine the criteria we can use to terminate an experiment. In this context, we hope that some of these biomarkers will be informative of mouse "clinical evolution" before the apparition of visible clinical signs. In this case the experiment could be terminated before the apparition of clinical sign for the mouse.
-Reduce the need for BM aspirates: As previously mentioned, the only way to conduct longitudinal studies with the primary AML model would be to perform sequential bone marrow aspiration from the femur or tibia using a needle. This is an effective but yet invasive approach. We propose here to assess the possibility to collect information from the bone marrow thanks to non-invasive in vivo imaging, which would represent a significant refinement of the experimental procedures.