Refining the lung cancer mouse model through environmental enrichment
Lead Research Organisation:
The Francis Crick Institute
Department Name: Research
Abstract
The five-year survival rate of patients with stage IV non-small cell lung cancer (NSCLC) is less than 10%, and only 15% of all lung cancer patients are alive 5 years after diagnosis (Ettinger et al. 2010). Immunotherapy is emerging as a viable treatment against late stage NSCLC (Rittmeyer et al. 2017 and Gandhi et al. 2018), but current lung cancer mouse models are unsuitable for studying the immune response to cancer (Mcfadden et al. 2016 and Dupage et al. 2013). In addition, many of the models that are used require large numbers of mice because of extensive breeding schemes (Dupage et al. 2011). Most mice in these experiments are housed in shoebox size containers with a maximum of 5 mice per cage with very little stimulation and no way for the mice to exercise. I aim to refine the lung cancer mouse model by studying if housing mice in enriched environments, which should improve their overall welfare, inhibits tumour growth, increases the anti-cancer immune response to more closely mimic what is observed in human tumours, and reduces cancer associated skeletal muscle loss known as cachexia.
To carry out this work, I will use a lung cancer transplant model where mice will be housed in an enriched environment for 5 weeks prior to transplant with a novel lung cancer cell line. The enriched environment will consist of running wheels, mouse igloos, wood blocks, nesting material, and tubing. The position and type of enrichment will be changed weekly to provide additional stimulation for the mice. Twenty mice will be housed together in each cage to increase social enrichment. Control mice will be housed in standard cages with 5 mice per cage.
To assess the welfare of the mice housed in control or enriched environments we will use an elevated plus maze to assess anxiety levels. In this maze there are two open arms and two closed arms and the more time the mice spend in the open arms of the tube indicates reduced levels of stress.
To determine if environmental enrichment and exercise reduces tumour growth, decreases cancer associated cachexia and boosts the anti-cancer immune response, tumour size, immune infiltrate, and muscle size of each mouse will be measured four weeks post-transplant. If environmental enrichment does boost the anti-cancer immune response in this model, it will also reduce the number of mice required to study the immune system's role in cancer as currently, extensive breeding schemes are required to generate the genetically engineered mouse models used to study the immune response to cancer, which would not be required for my model system.
Refining the lung cancer model is necessary for improving the wellbeing of the mice in these studies and is critical from a scientific perspective to understand the conditions associated with treatment response and improved outcomes. This refined lung cancer mouse model should uncover pathways that can potentially be targeted to combat patient relapse, cancer associated cachexia, and immune escape.
To carry out this work, I will use a lung cancer transplant model where mice will be housed in an enriched environment for 5 weeks prior to transplant with a novel lung cancer cell line. The enriched environment will consist of running wheels, mouse igloos, wood blocks, nesting material, and tubing. The position and type of enrichment will be changed weekly to provide additional stimulation for the mice. Twenty mice will be housed together in each cage to increase social enrichment. Control mice will be housed in standard cages with 5 mice per cage.
To assess the welfare of the mice housed in control or enriched environments we will use an elevated plus maze to assess anxiety levels. In this maze there are two open arms and two closed arms and the more time the mice spend in the open arms of the tube indicates reduced levels of stress.
To determine if environmental enrichment and exercise reduces tumour growth, decreases cancer associated cachexia and boosts the anti-cancer immune response, tumour size, immune infiltrate, and muscle size of each mouse will be measured four weeks post-transplant. If environmental enrichment does boost the anti-cancer immune response in this model, it will also reduce the number of mice required to study the immune system's role in cancer as currently, extensive breeding schemes are required to generate the genetically engineered mouse models used to study the immune response to cancer, which would not be required for my model system.
Refining the lung cancer model is necessary for improving the wellbeing of the mice in these studies and is critical from a scientific perspective to understand the conditions associated with treatment response and improved outcomes. This refined lung cancer mouse model should uncover pathways that can potentially be targeted to combat patient relapse, cancer associated cachexia, and immune escape.
Technical Summary
The 5-year survival rate of patients with stage IV non-small cell lung cancer is less than 10%(Ettinger et al. 2010). Although lung cancer mouse models have led to ground breaking findings in recent years, current models do not fully recapitulate the immune response and mutation burden observed in human tumors(Dupage et al. 2011; McFadden et al. 2016). Accumulating evidence suggests that housing mice in environmentally enriched environments has a variety of benefits(Pedersen et al. 2016; Song et al. 2017; Rampon et al. 2000; Benaroya-Milshtein et al. 2004; Kovesdi et al. 2011; Bice et al. 2017). In my proposal, I aim to refine the lung cancer mouse model by housing mice in an enriched environment to improve their well-being and assess if enriched housing inhibits tumour growth, increases the cancer immune response, and inhibits cancer associated cachexia.
Mice will be habituated to their environments for 5 weeks prior to intravenous injection with a novel immunogenic cell line KPAR1.3 derived from a tumour grown in a Rag-/-;KrasLSL-G12D;P53fl/fl;R26LSL-APOBEC3Bi mouse model of lung cancer. Anxiety will be assessed using an elevated plus maze. Tumour growth will be measured at 3 weeks post-injection by microCT, and at 4 weeks a grip test will be performed to determine cachexia levels. A subset of mice will be treated with checkpoint inhibitors to examine if tumours are more sensitive to immunotherapy in mice housed in enriched environments. Tumours will be analysed by immunohistochemistry and flow cytometry to analyse tumour immune infiltrate. A representative muscle in the mouse will be stained to assess muscle size to assess if cachexia is reduced with environmental enrichment.
This refined model system is easy to setup and applicable to many different cancers, so should have a broad impact across the cancer biology field. This model system should also reduce the number of mice required as complex breeding schemes are not necessary.
Mice will be habituated to their environments for 5 weeks prior to intravenous injection with a novel immunogenic cell line KPAR1.3 derived from a tumour grown in a Rag-/-;KrasLSL-G12D;P53fl/fl;R26LSL-APOBEC3Bi mouse model of lung cancer. Anxiety will be assessed using an elevated plus maze. Tumour growth will be measured at 3 weeks post-injection by microCT, and at 4 weeks a grip test will be performed to determine cachexia levels. A subset of mice will be treated with checkpoint inhibitors to examine if tumours are more sensitive to immunotherapy in mice housed in enriched environments. Tumours will be analysed by immunohistochemistry and flow cytometry to analyse tumour immune infiltrate. A representative muscle in the mouse will be stained to assess muscle size to assess if cachexia is reduced with environmental enrichment.
This refined model system is easy to setup and applicable to many different cancers, so should have a broad impact across the cancer biology field. This model system should also reduce the number of mice required as complex breeding schemes are not necessary.
Planned Impact
Using enriched housing, I aim to refine the lung cancer mouse model, which should improve both the welfare of the mice in, and the quality of the data produced from these studies. Current lung cancer mouse models do not fully recapitulate the tumour evolution or immune responses observed in human lung tumours (McFadden et al. 2016, Westcott et al. 2015, Chung et al. 2017, Dupage et al. 2013). Emerging evidence suggests that housing mice in enriched environments inhibits tumour growth through a variety of pathways including the induction of a more robust immune response (Pedersen et al. 2016, Song et al. 2017). I propose to study the mechanisms of tumour inhibition, immune response, and cachexia using a novel immunogenic lung cancer cell line transplanted into immunocompetent mice housed in an enriched environment. This will transform the field as no lung cancer mouse model exists that both significantly improves animal welfare while also more accurately modelling lung cancer evolution and the cancer immune response.
My refined lung cancer mouse model will also reduce the number of mice used as each mouse will produce more data, and extensive breeding schemes will not be necessary. In one study in Professor Tyler Jacks laboratory, which specifically aimed to study the T cell response in lung cancer (DuPage et al. 2011), each background strain was backcrossed 8 times to purify it, and extensive breeding was done to produce the genetically engineered mouse models necessary for the study. In this study, I estimate that approximately 400 mice were used. I know of 8 laboratories in the UK and the US including ours that use a similar number of mice for studies in their laboratories. Laboratories like ours run multiple studies at once, usually 3 to 4 a year, so approximately 1500 mice per year. This results in about 12,000 mice per year bred and housed under standard conditions amongst just the laboratories I know of. If successful my technique could reduce the number of mice in these laboratories from 12,000 to less than 5,000 a year, while also improving the welfare of the mice that are used. If my techniques are adopted, I believe a typical study would use about 200 mice, reducing the number to about 4,800 mice per year amongst the laboratories I know of. Using google scholar to search for the keywords: lung cancer mouse model and immune response since 2017 resulted in about 100 papers, suggesting there is a broader interest in studying the lung cancer immune response. If my model is successful other lung cancer studies where an immune response is important to the experiment could use my refined lung cancer model, reducing the number or mice required. The welfare of the mice used in these studies would also be significantly improved, with enriched housing, access to exercise wheels, and increased social stimulation.
In our laboratory at the Francis Crick Institute I am working with the biological research facility (BRF) to further refine my mouse model so we can measure the exact effects of each of the components of environmental enrichment. I also believe my model system is cheap and easy to implement making it more likely to be adopted by other laboratories.
I have contacted several Professors in the UK and the US, including Professor Martin McMahon at the Huntsman Institute, Professor Sergio Quezada at UCL, and Professor Reuben Harris at the University of Minnesota. They are all interested in adopting this housing system and have written letters stating their interest that are included in this application.
I believe this enriched housing system will have a significant impact on the cancer biology field because if successful it would improve both the science produced from, and the wellbeing of the mice in these experiments.
My refined lung cancer mouse model will also reduce the number of mice used as each mouse will produce more data, and extensive breeding schemes will not be necessary. In one study in Professor Tyler Jacks laboratory, which specifically aimed to study the T cell response in lung cancer (DuPage et al. 2011), each background strain was backcrossed 8 times to purify it, and extensive breeding was done to produce the genetically engineered mouse models necessary for the study. In this study, I estimate that approximately 400 mice were used. I know of 8 laboratories in the UK and the US including ours that use a similar number of mice for studies in their laboratories. Laboratories like ours run multiple studies at once, usually 3 to 4 a year, so approximately 1500 mice per year. This results in about 12,000 mice per year bred and housed under standard conditions amongst just the laboratories I know of. If successful my technique could reduce the number of mice in these laboratories from 12,000 to less than 5,000 a year, while also improving the welfare of the mice that are used. If my techniques are adopted, I believe a typical study would use about 200 mice, reducing the number to about 4,800 mice per year amongst the laboratories I know of. Using google scholar to search for the keywords: lung cancer mouse model and immune response since 2017 resulted in about 100 papers, suggesting there is a broader interest in studying the lung cancer immune response. If my model is successful other lung cancer studies where an immune response is important to the experiment could use my refined lung cancer model, reducing the number or mice required. The welfare of the mice used in these studies would also be significantly improved, with enriched housing, access to exercise wheels, and increased social stimulation.
In our laboratory at the Francis Crick Institute I am working with the biological research facility (BRF) to further refine my mouse model so we can measure the exact effects of each of the components of environmental enrichment. I also believe my model system is cheap and easy to implement making it more likely to be adopted by other laboratories.
I have contacted several Professors in the UK and the US, including Professor Martin McMahon at the Huntsman Institute, Professor Sergio Quezada at UCL, and Professor Reuben Harris at the University of Minnesota. They are all interested in adopting this housing system and have written letters stating their interest that are included in this application.
I believe this enriched housing system will have a significant impact on the cancer biology field because if successful it would improve both the science produced from, and the wellbeing of the mice in these experiments.
