A novel murine model of squamous lung cancer
Lead Research Organisation:
University of Cambridge
Department Name: Medicine
Abstract
Squamous lung cancer (SQC) is a type of non-small cell lung cancer (NSCLC). There are currently no specific therapeutic agents licensed to treat SQC and outcomes for patients with advanced disease are very poor. There are some targeted therapeutics available for the other common type of NSCLC - lung adenocarcinoma (ADC) - and more is understood about the molecular events that drive it.
Likewise, there are mouse models of lung adenocarcinoma available that have been very helpful in understanding its biology and that can have direct relevance to the clinical disease. A major advantage that mouse models have over human organoid approaches is that they can be used to understand the dynamic and complex relationship between the developing and progressing tumour and its microenvironment.
There is an urgent clinical need to improve our approach to SQC and this could be helped enormously by a rational mouse model that can replicate the genetic and tissue microenvironment context in which the tumour develops. Efforts to date have had some successes but, unfortunately, they are of limited utility in terms of translational studies or testing potential therapeutic combinations.
In order to address this unmet need we have developed a new way of making a mouse model of SQC. We use mouse cells from an animal's lung and expand them in tissue culture before manipulating them using laboratory techniques to recreate the genetic abnormalities seen in the human disease. We then inject these cells back into the lung of a genetically identical (inbred) mouse and these cells lead to SQC tumours.
This protocol has a major impact on the mission of the 3Rs. One reason is that for the traditional genetic models of this disease many breedings are required to get the right combination of genes to develop the type of cancer. In this case the genes are manipulated in cells rather than in an animal, so the process uses markedly fewer mice and is technically facile. A second reason is that we deliver the cells to one part of the lung, so that the animals develop a local tumour without becoming overtly unwell. This should allow us to refine the protocol so that the overall impact on animal welfare is significantly lessened. Third, many of the existing models of SQC use toxins to generate SQC with potentially complex and serious side effects. In this proposal we do not seek to administer toxins to any mouse at any stage. Finally we aim to engineer these cells so that we can monitor disease and the impact of a treatment in a single animal.
A further combined impact of these measures is a marked reduction in the cost of doing mouse experiments on SQC.
We have demonstrated in pilot data that our approach generates early murine SQC lesions within 3 weeks and large invasive tumours at around 4 months in wild type immunocompetent animals.
We aim to validate and reproduce our pilot data and demonstrate its direct relevance to the human disease. Further we will show that the model can be used efficiently to address key basic and translational issues: the necessity of a specific driving oncogene for the cancer cells to stay alive; the potential to use drugs to prevent squamous lung cancer development; and a demonstration that this model will have utility for studying how the tumour interacts with its microenvironment.
We are excited at the possibilties of this project and are enthusiastic about communicating with the potential end-users of this approach in academic community and pharma to ensure that the model is widely used.
Likewise, there are mouse models of lung adenocarcinoma available that have been very helpful in understanding its biology and that can have direct relevance to the clinical disease. A major advantage that mouse models have over human organoid approaches is that they can be used to understand the dynamic and complex relationship between the developing and progressing tumour and its microenvironment.
There is an urgent clinical need to improve our approach to SQC and this could be helped enormously by a rational mouse model that can replicate the genetic and tissue microenvironment context in which the tumour develops. Efforts to date have had some successes but, unfortunately, they are of limited utility in terms of translational studies or testing potential therapeutic combinations.
In order to address this unmet need we have developed a new way of making a mouse model of SQC. We use mouse cells from an animal's lung and expand them in tissue culture before manipulating them using laboratory techniques to recreate the genetic abnormalities seen in the human disease. We then inject these cells back into the lung of a genetically identical (inbred) mouse and these cells lead to SQC tumours.
This protocol has a major impact on the mission of the 3Rs. One reason is that for the traditional genetic models of this disease many breedings are required to get the right combination of genes to develop the type of cancer. In this case the genes are manipulated in cells rather than in an animal, so the process uses markedly fewer mice and is technically facile. A second reason is that we deliver the cells to one part of the lung, so that the animals develop a local tumour without becoming overtly unwell. This should allow us to refine the protocol so that the overall impact on animal welfare is significantly lessened. Third, many of the existing models of SQC use toxins to generate SQC with potentially complex and serious side effects. In this proposal we do not seek to administer toxins to any mouse at any stage. Finally we aim to engineer these cells so that we can monitor disease and the impact of a treatment in a single animal.
A further combined impact of these measures is a marked reduction in the cost of doing mouse experiments on SQC.
We have demonstrated in pilot data that our approach generates early murine SQC lesions within 3 weeks and large invasive tumours at around 4 months in wild type immunocompetent animals.
We aim to validate and reproduce our pilot data and demonstrate its direct relevance to the human disease. Further we will show that the model can be used efficiently to address key basic and translational issues: the necessity of a specific driving oncogene for the cancer cells to stay alive; the potential to use drugs to prevent squamous lung cancer development; and a demonstration that this model will have utility for studying how the tumour interacts with its microenvironment.
We are excited at the possibilties of this project and are enthusiastic about communicating with the potential end-users of this approach in academic community and pharma to ensure that the model is widely used.
Technical Summary
Squamous lung cancer (SQC) has been significantly more challenging to model than adenocarcinoma and progress in understanding its pathobiology and developing targeted therapeutics has been slow.
To address this unmet need we have developed a novel orthotopic model of SQC. We have refined/optimised a protocol for the expansion of primary murine tracheobronchial epithelial cells (mTBECs). We have optimised the genetic manipulation of primary mTBECs via multilocus CRISPR and lentiviral transduction so that clinically relevant genotypes can be rapidly recreated in primary mTBECs.
Further, we use inducible constructs so that genes of interest can be turned on and off at will throughout the natural history of the disease. The manipulated mTBECs are injected into the right lung of a syngeneic animal. Recipient mice therefore have an intact immune system and develop localised disease with the potential to metastasise.
We have demonstrated in pilot data that this approach generates early murine SQC lesions within 3 weeks and large invasive tumours at around 4 months in wild type immunocompetent animals.
This model can revolutionise the field - and lead to a dramatic reduction in the number of mice required to perform experiments; as well as refinements that reduce the ASPA Severity limit to Moderate and remove the need for toxin administration.
Our scientific aims are to characterise the natural history of SQC in this novel syngeneic murine orthotopic model, to validate the model as being both reproducible and directly relevant to the human disease. Further, we will use luciferase-based imaging to ensure longitudinal studies on individual animals are feasible. Second we will apply the model to efficiently address key basic and translational issues: the necessity of a driving oncogene for SQC maintenance; the potential to use AKT inhibition for SQC chemoprevention; and a demonstration that it will have utility for studying the tumour immune microenvironment.
To address this unmet need we have developed a novel orthotopic model of SQC. We have refined/optimised a protocol for the expansion of primary murine tracheobronchial epithelial cells (mTBECs). We have optimised the genetic manipulation of primary mTBECs via multilocus CRISPR and lentiviral transduction so that clinically relevant genotypes can be rapidly recreated in primary mTBECs.
Further, we use inducible constructs so that genes of interest can be turned on and off at will throughout the natural history of the disease. The manipulated mTBECs are injected into the right lung of a syngeneic animal. Recipient mice therefore have an intact immune system and develop localised disease with the potential to metastasise.
We have demonstrated in pilot data that this approach generates early murine SQC lesions within 3 weeks and large invasive tumours at around 4 months in wild type immunocompetent animals.
This model can revolutionise the field - and lead to a dramatic reduction in the number of mice required to perform experiments; as well as refinements that reduce the ASPA Severity limit to Moderate and remove the need for toxin administration.
Our scientific aims are to characterise the natural history of SQC in this novel syngeneic murine orthotopic model, to validate the model as being both reproducible and directly relevant to the human disease. Further, we will use luciferase-based imaging to ensure longitudinal studies on individual animals are feasible. Second we will apply the model to efficiently address key basic and translational issues: the necessity of a driving oncogene for SQC maintenance; the potential to use AKT inhibition for SQC chemoprevention; and a demonstration that it will have utility for studying the tumour immune microenvironment.
Planned Impact
This proposal concerns the mouse - the most common animal used for research into cancer pathobiology or therapeutics. Successful completion of this project will address two of the three core missions of the NC3Rs - reduction and refinement.
It will dramatically reduce the number of mice used to generate experimental mice for research/therapeutic testing in SQC. Second, it will remove the need to use toxins to generate mouse models of SQC - therefore reducing the adverse welfare impacts resulting from toxin use. Third, it will reduce the severity level for modelling SQC from Severe to Moderate.
This project will develop, validate and provide example applications for a new murine model of SQC for the academic and industrial community. It should be adopted widely as it is straightforward to replicate, does not necessitate complex breeding protocols, has multiple aspects that mean it is extremely cost effective, and we have developed a comprehensive dissemination plan detailed in the Pathways statement.
Reduction
There are two types of murine models of SQC - toxin-induced and genetically engineered models (GEMMs). The best/most clinically relevant current GEMM necessitates a double homozygous genotype crossed with a Sox2 transgene as well as intraperitoneal naphthalene (see below). Using our protocol which requires no specialised breeding protocols, there would therefore be a reduction of well over 80% in the number of mice required to generate experimental animals. Over the last few years our mice numbers have averaged approximately 900/yr. To date, in this calendar year, having changed to this protocol we have used a total of 37 mice so that it has already had a significant impact locally.
In the UK there are at least 4 laboratories investigating SQC using up to 4000 animals per year.
We estimate (conservatively) that the impact of the adoption of our protocol is a reduction of 80% or 3200 mice. Globally the number of animals impacted is more difficult to quantify, but we estimate that at least 20,000 mice per annum are used in SQC research. This is based on Pubmed-listed publications from fourteen laboratories in the last 5 years using GEMMs or toxin-induced models of SQC and on personal knowledge of current activity in at least 4 other major national/international academic or pharma laboratories.
If this protocol were adopted widely then we estimate the absolute reduction in mouse numbers would be in the region of 10,000. This estimate reflects the fact that the toxin (NTCU) model is currently used in more laboratories than GEMMs.
Refinement
Currently the severity limit for our models is Severe. A reduction in severity is a key aim of this proposal. Importantly, only one mouse in the pilot cohort (n=37) had Mild clinical concerns prior to culling. This likely reflects the limited involvement of one lung in this model rather than the multilobar bilateral involvement typical of mouse models of SQC. Given the absence of overt clinical signs/significant weight loss we are confident that this protocol will result in a reduction in severity from Severe to Moderate.
A second major refinement is that mice will no longer need to be treated with toxins to generate the desired phenotype. Multiple groups worldwide use the NTCU model to generate SQC lesions. This requires the repeated administration of the toxin to skin twice a week over a period of up to 32 weeks. The protracted administration and the need to use a dose with likely systemic toxicity (See Pathways to Impact) has a significant impact on animal welfare. The best current GEMM use intraperitoneal naphthalene to denude airway epithelium and provoke injury and repair - a procedure that also has considerable potential implications for animal welfare (See Pathways to Impact). In the current proposal we remove the need to use toxins to generate murine models of SQC.
Taken together these measures will have a marked impact on the meeting the 3Rs mission.
It will dramatically reduce the number of mice used to generate experimental mice for research/therapeutic testing in SQC. Second, it will remove the need to use toxins to generate mouse models of SQC - therefore reducing the adverse welfare impacts resulting from toxin use. Third, it will reduce the severity level for modelling SQC from Severe to Moderate.
This project will develop, validate and provide example applications for a new murine model of SQC for the academic and industrial community. It should be adopted widely as it is straightforward to replicate, does not necessitate complex breeding protocols, has multiple aspects that mean it is extremely cost effective, and we have developed a comprehensive dissemination plan detailed in the Pathways statement.
Reduction
There are two types of murine models of SQC - toxin-induced and genetically engineered models (GEMMs). The best/most clinically relevant current GEMM necessitates a double homozygous genotype crossed with a Sox2 transgene as well as intraperitoneal naphthalene (see below). Using our protocol which requires no specialised breeding protocols, there would therefore be a reduction of well over 80% in the number of mice required to generate experimental animals. Over the last few years our mice numbers have averaged approximately 900/yr. To date, in this calendar year, having changed to this protocol we have used a total of 37 mice so that it has already had a significant impact locally.
In the UK there are at least 4 laboratories investigating SQC using up to 4000 animals per year.
We estimate (conservatively) that the impact of the adoption of our protocol is a reduction of 80% or 3200 mice. Globally the number of animals impacted is more difficult to quantify, but we estimate that at least 20,000 mice per annum are used in SQC research. This is based on Pubmed-listed publications from fourteen laboratories in the last 5 years using GEMMs or toxin-induced models of SQC and on personal knowledge of current activity in at least 4 other major national/international academic or pharma laboratories.
If this protocol were adopted widely then we estimate the absolute reduction in mouse numbers would be in the region of 10,000. This estimate reflects the fact that the toxin (NTCU) model is currently used in more laboratories than GEMMs.
Refinement
Currently the severity limit for our models is Severe. A reduction in severity is a key aim of this proposal. Importantly, only one mouse in the pilot cohort (n=37) had Mild clinical concerns prior to culling. This likely reflects the limited involvement of one lung in this model rather than the multilobar bilateral involvement typical of mouse models of SQC. Given the absence of overt clinical signs/significant weight loss we are confident that this protocol will result in a reduction in severity from Severe to Moderate.
A second major refinement is that mice will no longer need to be treated with toxins to generate the desired phenotype. Multiple groups worldwide use the NTCU model to generate SQC lesions. This requires the repeated administration of the toxin to skin twice a week over a period of up to 32 weeks. The protracted administration and the need to use a dose with likely systemic toxicity (See Pathways to Impact) has a significant impact on animal welfare. The best current GEMM use intraperitoneal naphthalene to denude airway epithelium and provoke injury and repair - a procedure that also has considerable potential implications for animal welfare (See Pathways to Impact). In the current proposal we remove the need to use toxins to generate murine models of SQC.
Taken together these measures will have a marked impact on the meeting the 3Rs mission.
