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IAT Congress poster final

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Paul Mackin

The document describes using ultrasound imaging to monitor tumor growth in a mouse model of pancreatic cancer. Mice are palpated weekly to identify tumors, which are then measured via ultrasound if between 2-9mm in size. Mice with 6-9mm tumors are enrolled in therapeutic studies and scanned twice weekly to quantify tumor volumes over time and evaluate drug responses. The non-invasive ultrasound technique allows accurate, repetitive measurement of tumor size and growth rates under investigation drugs with minimal stress to the mice.

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Cage to bedside: Ultrasound imaging in a mouse model of Pancreatic ductal adenocarcinoma (PDA) Paul Mackin, Lisa Young, Frances Connor, David Tuveson. Cancer Research UK, Cambridge Research Institute, Cambridge. UK Correspondence: paul.mackin@cancer.org.uk Pancreatic ductal adenocarcinoma (PDA) is the fifth most common cause of cancer death in the UK causing 7,781 deaths in 2008. Prognosis is extremely poor; survival of patients with advanced disease is about 6 months. Despite major efforts in basic and clinical research, most therapies still fail to improve survival and novel therapies are urgently required for this fatal disease. As tumour onset cannot be predicted, all KPC mice (from the age of 2 months) are abdominally palpated weekly to identify the onset of tumour presence, being graded by palpable size (*2-3mm, ** 4-6mm *** 7- 9mm ). During these weeks mice are closely monitored and any showing clinical signs of ill health are either used on short term studies or sacrificed for blood, tissue samples or both. We are refining the assessment of pre-enrolable tumours by precise palpations and reducing the stress of mice being repeatedly ultrasound scanned to follow the growth pattern of the tumours as well as the associated implications of repeated exposure to anaesthetic. 0 100 200 300 400 0 20 40 60 80 100 KPC PC Time PercentsurvivalThe Tuveson lab has produced a genetically engineered mouse model (KPC) carrying Kras and p53 mutations that recapitulate the clinical and pathological features of human disease including site of occurrence, tumours, metastasis, tumour microenvironment, haemorrhagic ascites etc. This model is therefore likely to be more predictive of response to novel therapies that can then be translated to patients. The median survival of the KPC model is 5.6 months, although the latency varies with mice developing tumours between 2 and 8 months. Mice which are developing ** to *** tumours are scheduled for ultrasound scanning to establish tumour location and confirmation of size. Once measured at 6- 9mm diameter they are enrolled onto therapeutic study and twice weekly ultrasounds are scheduled to quantify tumour volume over time. This allows us to evaluate the effects of drugs on tumour growth. 12.00 3.00 6.00 9.00 Mouse Orientation Probe Orientation We do know that as the tumour develops (to an average dimension of 6-9mm at its widest point) the growth curve accelerates quite rapidly. This is the ideal time to begin therapy, as we will see either 1. continued tumour development, 2. flattening of the growth curve, 3. decrease in tumour size over time. Acknowledgements to the Mouse Hospital Team Steve Kupczak Maureen Cronshaw Lauren Shelbourne, Yi Cheng In the past allograft and xenograft models have used countless animals to show therapeutic efficacy, with such results very rarely translating to positive outcome in the clinic. In contrast, we believe that the KPC model is a better predictor of response to therapy. Indeed, we have shown that this model responds to Gemcitabine, the standard of care for pancreatic cancer, in a similar manner as seen in the clinical setting. To date, over ten drugs have been tested in the Mouse Hospital. Studies highlighting the barrier to drug delivery by the excessive stroma are of particular interest. We have shown that the efficacy of chemotherapy can be improved by the depletion of tumour-associated stroma, resulting in an extended lifespan These promising results are currently translated into clinical practice; two clinical trials have recently opened to investigate experimental drugs from the Mouse Hospital either in advanced or pre-operative PDA patients. We use the Visualsonics Vevo 2100 ultrasound machine with 13 - 55 MHz probes enabling clear image acquisition. To facilitate imaging, hair is removed by shaving and depilatory cream. During imaging, mice are placed on a heated platform. A maximum of 4ml sterile saline is injected intraperitoneally to suspend the internal organs. The mouse may need to be rotated to give optimum vision of the tumour. Pancreatic Ductal Adenocarcinoma The KPC model Tumour identification by palpation Ultrasound Imaging of tumours Enrolment of mice on therapeutic studies The utility of the KPC model Study US Birth Palpable Tumour Enrolment 6 - 9 mm Disease Free Weekly US 2 mths ~ 5.5 mths Study US Study US Study US Survival - 10 days no treatment Therapeutic study Therapeutic intervention – Survival??? Pre-enrolment Quantifying tumour volume Probe Orientation Why different angles? Mouse Pancreas Anatomy Bile Duct Spleen L. Kidney R. Kidney Bladder Stomach Duo Head Tail Body Arm Panc. Duct CBD ULTRASOUND IMAGING PANCREATIC TUMOURS Tumour size accurately measured using ultrasound -18 -12 -6 0 6 12 0 100 200 300 400 500 600 Growth of tumour %InitialVolume 3D reconstructions of tumour taken at five time points
Mice are palpated weekly for tumour presence; 1 to 3 mm tumours can generally be felt. Once confirmed, dependant on size, tumours are either re-palpated or scheduled for ultrasound imaging to follow tumour growth. However, recognising that repeated anaesthesia is a stressor, we have been working to increase the precision of our palpations before scanning begins. We have successfully trained the animal facility staff to palpate accurately, we are able to identify tumours at the enrolable size and so again reduce the number of pre enrolment scans before entering therapeutic study. Mice are enrolled onto study once tumours are 6 to 9 mm in diameter. Each animal is scanned from at least 2 or 3 different angles as the pancreas in mice is relatively diffuse in shape (unlike the human) it’s important that tumours are not missed. Fur from the abdomen is removed under general anaesthesia, using a commercially available depilatory cream, the day of scanning. Saline is given IP to enable the internal organs and tumour to ‘float’ freely, allowing separation between organs - better view - better quality imaging. When using the older Vevo 770 needed to administer around 3-4ml (as authorised in our PPL), but since upgrading to Vevo 2100 we can acquire very clear images with good separation using approx. 2mls or none! Again, using the Vevo 2100, because the images are now so much clearer we acquire them in a shorter time, therefore the mice are under anaesthetic for less time and recovery time is shorter. After scanning mice are cleaned of imaging gel before being place in a heated recovery chamber, they are then monitored for righting reflex and mobility before being put back in their home cage, with a ‘Scanned’ card stating mouse ID and date scanned. Scanned mice are physically checked the next day to ensure diuresis and full recovery from the ultrasound procedure Ability to build up a detailed, three dimensional picture of each tumour and quantify its volume over time. Therapies can be evaluated more accurately than before using this technique, and humane endpoints implemented with greater precision. Using the Vevo 770 we would routinely schedule 4 scan sessions per week (Wed am, Wed pm, Thur am Thur pm. 10-12 mice per session, with each session lasting approx. 3.5 hrs. Today with the new Vevo 2100 we now routinely scan 2 sessions on one day per week but still maintaining the number of mice approx. 25-35 only occasionally adding a 3rd session. Summary

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IAT Congress poster final

  • 1. Cage to bedside: Ultrasound imaging in a mouse model of Pancreatic ductal adenocarcinoma (PDA) Paul Mackin, Lisa Young, Frances Connor, David Tuveson. Cancer Research UK, Cambridge Research Institute, Cambridge. UK Correspondence: paul.mackin@cancer.org.uk Pancreatic ductal adenocarcinoma (PDA) is the fifth most common cause of cancer death in the UK causing 7,781 deaths in 2008. Prognosis is extremely poor; survival of patients with advanced disease is about 6 months. Despite major efforts in basic and clinical research, most therapies still fail to improve survival and novel therapies are urgently required for this fatal disease. As tumour onset cannot be predicted, all KPC mice (from the age of 2 months) are abdominally palpated weekly to identify the onset of tumour presence, being graded by palpable size (*2-3mm, ** 4-6mm *** 7- 9mm ). During these weeks mice are closely monitored and any showing clinical signs of ill health are either used on short term studies or sacrificed for blood, tissue samples or both. We are refining the assessment of pre-enrolable tumours by precise palpations and reducing the stress of mice being repeatedly ultrasound scanned to follow the growth pattern of the tumours as well as the associated implications of repeated exposure to anaesthetic. 0 100 200 300 400 0 20 40 60 80 100 KPC PC Time PercentsurvivalThe Tuveson lab has produced a genetically engineered mouse model (KPC) carrying Kras and p53 mutations that recapitulate the clinical and pathological features of human disease including site of occurrence, tumours, metastasis, tumour microenvironment, haemorrhagic ascites etc. This model is therefore likely to be more predictive of response to novel therapies that can then be translated to patients. The median survival of the KPC model is 5.6 months, although the latency varies with mice developing tumours between 2 and 8 months. Mice which are developing ** to *** tumours are scheduled for ultrasound scanning to establish tumour location and confirmation of size. Once measured at 6- 9mm diameter they are enrolled onto therapeutic study and twice weekly ultrasounds are scheduled to quantify tumour volume over time. This allows us to evaluate the effects of drugs on tumour growth. 12.00 3.00 6.00 9.00 Mouse Orientation Probe Orientation We do know that as the tumour develops (to an average dimension of 6-9mm at its widest point) the growth curve accelerates quite rapidly. This is the ideal time to begin therapy, as we will see either 1. continued tumour development, 2. flattening of the growth curve, 3. decrease in tumour size over time. Acknowledgements to the Mouse Hospital Team Steve Kupczak Maureen Cronshaw Lauren Shelbourne, Yi Cheng In the past allograft and xenograft models have used countless animals to show therapeutic efficacy, with such results very rarely translating to positive outcome in the clinic. In contrast, we believe that the KPC model is a better predictor of response to therapy. Indeed, we have shown that this model responds to Gemcitabine, the standard of care for pancreatic cancer, in a similar manner as seen in the clinical setting. To date, over ten drugs have been tested in the Mouse Hospital. Studies highlighting the barrier to drug delivery by the excessive stroma are of particular interest. We have shown that the efficacy of chemotherapy can be improved by the depletion of tumour-associated stroma, resulting in an extended lifespan These promising results are currently translated into clinical practice; two clinical trials have recently opened to investigate experimental drugs from the Mouse Hospital either in advanced or pre-operative PDA patients. We use the Visualsonics Vevo 2100 ultrasound machine with 13 - 55 MHz probes enabling clear image acquisition. To facilitate imaging, hair is removed by shaving and depilatory cream. During imaging, mice are placed on a heated platform. A maximum of 4ml sterile saline is injected intraperitoneally to suspend the internal organs. The mouse may need to be rotated to give optimum vision of the tumour. Pancreatic Ductal Adenocarcinoma The KPC model Tumour identification by palpation Ultrasound Imaging of tumours Enrolment of mice on therapeutic studies The utility of the KPC model Study US Birth Palpable Tumour Enrolment 6 - 9 mm Disease Free Weekly US 2 mths ~ 5.5 mths Study US Study US Study US Survival - 10 days no treatment Therapeutic study Therapeutic intervention – Survival??? Pre-enrolment Quantifying tumour volume Probe Orientation Why different angles? Mouse Pancreas Anatomy Bile Duct Spleen L. Kidney R. Kidney Bladder Stomach Duo Head Tail Body Arm Panc. Duct CBD ULTRASOUND IMAGING PANCREATIC TUMOURS Tumour size accurately measured using ultrasound -18 -12 -6 0 6 12 0 100 200 300 400 500 600 Growth of tumour %InitialVolume 3D reconstructions of tumour taken at five time points
  • 2. Mice are palpated weekly for tumour presence; 1 to 3 mm tumours can generally be felt. Once confirmed, dependant on size, tumours are either re-palpated or scheduled for ultrasound imaging to follow tumour growth. However, recognising that repeated anaesthesia is a stressor, we have been working to increase the precision of our palpations before scanning begins. We have successfully trained the animal facility staff to palpate accurately, we are able to identify tumours at the enrolable size and so again reduce the number of pre enrolment scans before entering therapeutic study. Mice are enrolled onto study once tumours are 6 to 9 mm in diameter. Each animal is scanned from at least 2 or 3 different angles as the pancreas in mice is relatively diffuse in shape (unlike the human) it’s important that tumours are not missed. Fur from the abdomen is removed under general anaesthesia, using a commercially available depilatory cream, the day of scanning. Saline is given IP to enable the internal organs and tumour to ‘float’ freely, allowing separation between organs - better view - better quality imaging. When using the older Vevo 770 needed to administer around 3-4ml (as authorised in our PPL), but since upgrading to Vevo 2100 we can acquire very clear images with good separation using approx. 2mls or none! Again, using the Vevo 2100, because the images are now so much clearer we acquire them in a shorter time, therefore the mice are under anaesthetic for less time and recovery time is shorter. After scanning mice are cleaned of imaging gel before being place in a heated recovery chamber, they are then monitored for righting reflex and mobility before being put back in their home cage, with a ‘Scanned’ card stating mouse ID and date scanned. Scanned mice are physically checked the next day to ensure diuresis and full recovery from the ultrasound procedure Ability to build up a detailed, three dimensional picture of each tumour and quantify its volume over time. Therapies can be evaluated more accurately than before using this technique, and humane endpoints implemented with greater precision. Using the Vevo 770 we would routinely schedule 4 scan sessions per week (Wed am, Wed pm, Thur am Thur pm. 10-12 mice per session, with each session lasting approx. 3.5 hrs. Today with the new Vevo 2100 we now routinely scan 2 sessions on one day per week but still maintaining the number of mice approx. 25-35 only occasionally adding a 3rd session. Summary