Impact of targeting myeloid PI3Kg on the pancreatic tumour stroma compartment and the response to chemotherapy.

Lead Research Organisation: University of Liverpool
Department Name: Institute of Translational Medicine

Abstract

Pancreatic cancer is very aggressive and kills around 8000 people every year in the UK. New treatment strategies are urgently needed. During tumour (cancer) formation, cancer cells attract cells from the immune system (white blood cells), which progressively accumulate in the tumour environmental bed (or stroma), producing an appearance of inflammation. Although it was originally thought that tumour infiltrating immune cells would kill the cancer cells, recent evidence suggests that they may actually promote tumour growth. In the tumour stroma certain immune cells, secrete high amounts of stimulatory factors that aid the cancer cells to multiply, survive, and spread. The abundance of white blood cells may determine whether the patient will die of the cancer or not.

The tumour infiltrating white blood cells still have the capability to kill cancer cells, but their anti-cancer functions are inhibited by cancer cells themselves and are skewed towards cancer promoting functions. Our preliminary results indicate that blockade of a specific molecule (known as PI3K gamma) present in certain white cells called macrophages, inhibits their cancer promoting functions and restores their initial cancer cell killing ability. Since macrophages are critical players in regulating the immune response, studying and targeting this recently identified function of PI3K gamma represents a promising approach to re-activate the immune system to fight against cancer.

The pancreatic cancer stroma comprises of two other types of normally resident cells known as pancreatic stellate cells and fibroblasts. These cells are responsible for secreting a high amount of substances called extracelluar matrix proteins that provide the scaffolding (or matrix) for the shape of the tissue and also the glue to hold the cells to the scaffolding. This is the stroma. In pancreatic cancer there is far too much of this stroma which means that anti-cancer drugs cannot properly reach the cancer cells to kill them.

Our early work shows that in pancreatic cancer patients, macrophages are found side by side with stellate cells, indicating a potential interaction between these cell types. Since we are able to change macrophage function by targeting PI3K gamma, we are now in the position to use this tool to study a so far uncharacterized interaction of stellate cells with macrophages.

Therefore, these studies will increase our knowledge concerning the cancer promoting functions of tumour associated macrophages and might reveal new key proteins that regulate their function and contribute to the poor response to chemotherapy. Our findings will also open new strategies to improve the treatments of pancreatic cancer.

The project proposed here focuses on the role of the host immune cells during pancreatic cancer progression and its response to chemotherapy. While most research has so far focused on the cancer cells themselves and their precursor cells, it has become clear that the immune system and the formation of a tumour microenvironment also influences the course of pancreatic cancer progression and its response to chemotherapy.

I believe that the here proposed project addresses important emerging questions in the pancreatic cancer field and that these studies will contribute to increase the understanding of how tumour inflammation impacts on pancreatic cancer progression.

This will help lead to better diagnosis and better treatments.

Technical Summary

Pancreatic ductal adenocarcinoma (PDA) has one of the highest mortality rates of any malignancy and effective treatment is major unmet need. A marked infiltration of myeloid cells into the stromal compartment and the generation of a desmoplastic stromal reaction is a particular characteristic of PDA and is thought to play a key role in disease progression and the poor response to therapy.Consequently, there is an urgent need to develop effective therapies targeting the stromal compartment in PDA.

Aim:
The proposed study aims to identify and target myeloid signalling pathways that promote pro-tumourigenic myeloid cell functions and desmoplasia in pancreatic cancer.

Objectives and planned methodologies:
This study will:

1. Use pre-clinical pancreatic tumour models in combination with genetic tools to inhibit gene expression (e.g. PI3Kg) in tumour associated macrophages.

2. Generate and use primary pancreatic stellate cells and primary macrophages in co-culture assays to characterise the interaction between these two stromal cell types.

3. Use a proteomic approach to identify PI3Kg downstream effectors regulating macrophage conversion, tumour immunity, PSC activation and desmoplasia.

4. Evaluate identified factors in vitro and in vivofor their impact on tumour immunity and desmoplasia formation.

5. Test in pre-clinical mouse PDA models whether blockade of PI3Kg and/or the newly identified downstream effectors in combination with standard chemotherapy (gemcitabine or 5FU) improves therapeutic outcome.

Results:
The preclinical testing of potential therapies will identify treatment options that can be translated into clinical trials. In using both genetic and pharmacological approaches, this programme of work maximises the likelihood of successfully identifying specific novel targets that are necessary for the tumour promoting functions of macrophages and provide the rationale for developing new anti-cancer therapeutic approaches.

Planned Impact

The Pancreas Research Group in Liverpool concentrates on investigating fundamental cellular mechanisms relevant to the pathophysiology of acute pancreatitis and pancreatic ductal adenocarcinoma (PDA) and translation into clinical outputs. The work of our team is closely co-ordinated through the Centre of Excellence for Pancreas Cancer of the Liverpool Cancer Research UK Centre and the Liverpool NIHR Pancreas Biomedical Research Unit, which is the only NIHR Research Unit focused on translational pancreatic research. The MRC funding requested in this application will further underpin conceptual and technical developments in the Pancreas Research Group necessary for translational progression. This Group coordinates the efforts of clinicians, cell biologists and physiologists, organic chemists, structural biologists and pharmacologists of the University of Liverpool aiming to develop drugs against PDA (as well as pancreatitis).

In the framework of this project, all information and knowledge acquired will be immediately shared with the Pancreatic Research Group and could be used for the informed design of pharmacological approaches for the treatment of PDA. All technical information and cell-based assays performed will also be shared with the Pancreatic Research Group and could be utilised for the development of procedures to test new pharmacological compounds in pancreatic cancer patients. The Pancreatic Research Group has established contacts with pharmaceutical companies interested in developing or testing compounds which could alleviate PDA. The relevant conceptual knowledge and methodological expertise generated in the course of our project will be conveyed to collaborating pharmaceutical companies. Collaborators are also actively involved in communicating the results of their research to the general public (see Pathways to Impact).

The advanced equipment (e.g. multicolour flow cytometry, in vivo imaging techniques, state-of-the-art Aperio Technologies Slide Scanning System for TMA analysis) available in the Pancreas Research Group and the technical expertise accumulated in the group (e.g. vast experience in staining, scoring and interpreting data from TMAs with assistance from pancreas histopathologists, and the isolation and culturing of primary human and murine pancreatic cancer and stellate cells) will complement high throughput instruments and techniques established in the NIHR unit to create an optimal technology platform, necessary for both, discovery and translation. Importantly, the Pancreas Research Group serves as a training base for MRC, Wellcome Trust and Cancer Research UK PhD students, as well as for clinical fellows undertaking research projects.

The MRC support will allow me to establish my team among the existing pancreatic research teams here, within the Liverpool Pancreas Research Group and my expertise and interest in the tumour microenvironment of PDA will ideally complement existing research efforts. The postdoctoral research scientist funded by this application and myself, will strongly be involved in the collaboration with the Liverpool Cancer Research UK Centre and the Liverpool NIHR Pancreas Biomedical Research Unit. The MRC support for the salary of an experienced postdoctoral scientist will also allow the establishment and maintenance of technical skills and transfer of expertise in advanced experimentation to future PhD students.

The MRC support of the current grant application will therefore not only provide resources necessary for the specific project, aiming to characterise crucial interactions of stromal cells in PDA disease progression, but will also enhance the collaborative efforts and the translational research of the Pancreas Research Group and strengthen the training base for PhD students with an interest in tumour inflammation and immunity, cell biology and in vivo tumour models of PDA.

Publications


10 25 50
 
Description NWCR - Doctoral Training Programme
Amount £140,000 (GBP)
Organisation North West Cancer Research (NWCR) 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 09/2014 
End 09/2018
 
Description Project Grant
Amount £160,000 (GBP)
Organisation Pancreatic Cancer Research Fund 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 08/2015 
End 07/2018
 
Description The role of macrophage-derived granulin in pancreatic cancer metastasis - mechanisms and therapeutic opportunities
Amount £850,000 (GBP)
Funding ID MR/P018920/1 
Organisation MRC-UK 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start  
 
Title Establishment of hepatic stellate cell cultures 
Description In collaboration with Dr Sakai Takeo's group at our institute, we have established the isolation of primary hepatic stellate cells from the liver. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact We are now using this method to generate freshly isolated hepatic stellate cells to study their interaction in response to cancer cell derived factors. 
 
Title Establishment of in vivo imagaing models 
Description We were able to implement state of the art in vivo imaging technology into our research studies. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact We are now in the position to monitor tumour progression and response to treatments in vivo over time. 
 
Title Establishment of murine primary pancreatic cancer cell lines 
Description We have established a panel of primary pancreatic murine cells lines derived from the spontaneous pancreatic tumour model Kras; p53, Pdx1-Cre. These cells lines harbor the most frequent genetic mutations driving pancreatic cancer progression (i.e. Kras and p53). These low passage primary cells show distinct biological responses compared to established cell lines which better represents the in vivo situation. Thus, working with these cell cultures critically improves the relevance of our in vitro cell culture assays. 
Type Of Material Cell line 
Year Produced 2014 
Provided To Others? Yes  
Impact These low passage primary cells show distinct biological responses compared to established cell lines. These cells respond in similar ways as observed in in vivo studies. Thus, working with these cell cultures critically facilitates the interpretation of results obtained from in vitro assays and consequently increases their impact. 
 
Title Establishment of murine primary pancreatic fibroblast cell lines 
Description We have established methods to isolate primary fibroblasts and stellate cells from pancreatic tumours and normal control tissue. 
Type Of Material Cell line 
Year Produced 2014 
Provided To Others? Yes  
Impact These methods allow us to study the cross-talk of myofibroblasts with different cell types present in the pancreatic tumour microenvironment (including cancer cells, immune cells). 
 
Title Establishment of transduction methods using lentiviral vectors 
Description We have established methods to transduce ex-vivo primary murine hematopoietic cells with lentiviral vectors. Furthermore, we have established methods to generate chimeric mice carrying the transduced hematopoietic cells. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2014 
Provided To Others? Yes  
Impact This method will allow us to genetically target gene expression and/or to introduce a reporter gene into hematopoietic cells in vivo and to analyse impact on disease progression in vivo. 
 
Title Experimental metastasis models 
Description We have established two experimental metastasis mouse models for pancreatic cancer. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact We are using the established experimental metastasis model to study the role of macrophages in pancreatic cancer metastasis. Using these models, we have generated a critical amount of data which is part of a manuscript currently under revision. 
 
Title MRI Imaging 
Description We have established methods and protocols to measure therapy response of metastatic tumour burden in living mice by MRI technology. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact We have shared our acquired expertise with other research groups within the institute. This enhanced in the quality and accuracy of our research. 
 
Title Mass cytometry 
Description We are currently establishing methods and protocols to use mass cytometry to study the role of immune cells, specifically macrophages, in cancer. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact Using this new technology, we are now able to study in more depth immune responses during cancer progression and in response to chemotherapeutic intervention. 
 
Title Orthotopic pancreatic cancer tumour models 
Description Orthotopic implantation of primary pancreatic tumour cells derived from tumour bearding KPC mice (KrasG12D;Trp53R172H) into syngeneic mice. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2014 
Provided To Others? Yes  
Impact This model represents an attractive alternative to the genetic engineered KPC mouse models (GEMM) of pancreatic cancer, while orthotopic implanted pancreatic tumors still resemble pathophysiological and molecular features of human PDA. However, this KPC-based orthotopic model is cost effective, tumour progression occurs with only small variations, and it is compatible with in vivo imaging techniques. This in vivo model allows us to study the impact of pharmacological intervention on pancreatic cancer progression. 
 
Description Crispr/Cas9 gene editing 
Organisation Korea Research Institute of Bioscience and Biotechnology (KRIBB)
Country Korea, Republic of 
Sector Academic/University 
PI Contribution We have established several murine pancreatic cancer cells lines. We are now interested to test whether the expression of programmed death ligand 1 (PD-L1), a ligand for the immune checkpoint receptor PD-1, impacts metastatic progression of these cancer cells. Thus, our collaborator at the KRIBB will deplete PD-L1 expression on our cell lines and send them back to our lab. We will next test the impact of PD-L1 expression on pancreatic cancer cell in on pancreatic cancer metastasis.
Collaborator Contribution Perform gene editing using Crispr/Cas9 based methodologies to deplete PD-L1 expression on our established murine cell lines.
Impact Pending
Start Year 2017
 
Description Fresh human liver tissue analysis 
Organisation Royal Liverpool and Broadgreen University Hospitals NHS Trust
Department Royal Liverpool University Hospital
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Hospitals 
PI Contribution Based on our recent research findings in pre-clinical mouse models, we have now arrange a new collaboration with clinicians at the Liverpool Royal Hospital and Aintree Hospital. My group currently establishing methods and protocols to analyze by flow and mass cytometry fresh liver tissue biopsies from metastatic pancreatic cancer patients. Having access to fresh metastatic liver tissue samples will allow us to compare our findings obtained in mouse models in human patient samples.
Collaborator Contribution Our partners, including clinicans and research nurses, are arranging the necessary ethical approvals and arrange the patient consent forms. Biopsies are taken as part of the routine diagnosis and are immediatly handed over to our research staff.
Impact We are currently in the process of establishing methods and protocols allowing us in future to analyze fresh human tissue samples by mass and flow cytometry.
Start Year 2017
 
Description Human tissue sections and TMA 
Organisation University of Liverpool
Department Molecular and Clinical Cancer Medicine Liverpool
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Staining and analysing of human tissue sections and TMA
Collaborator Contribution Providing access to stored human tissue and TMA
Impact N/A
Start Year 2013
 
Description KPC model 
Organisation Cold Spring Harbor Laboratory (CSHL)
Department Cancer Centre
Country United States of America 
Sector Academic/University 
PI Contribution Import of the genetic engineered mouse model of pancreatic cancer (KPC animals). Establishment of primary pancreatic cancer cell lines derived tumour bearing KPC animals. Establishment of primary cancer associated fibroblast (CAFs) cultures from tumour bearing KPC animals
Collaborator Contribution Transfer of KPC mice to Univeristy of Liverpool
Impact N/A
Start Year 2013
 
Description LV shRNA system 
Organisation University of Edinburgh
Department The Roslin Institute
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Constructing of a lentivirus for shRNA mediated knockdown in monocytes
Collaborator Contribution Providing backbone for lentivirus, including monocyte specific promoter sequence
Impact N/A
Start Year 2013
 
Description Methods to study hepatic stellate cell activation and migration 
Organisation University of Liverpool
Department Department of Molecular and Clinical Pharmacology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Our team has set up several in vitro assays to quantify the activation of primary hepatic stellate cells in response to stimulatory factors.
Collaborator Contribution Dr Sakai's research group has provided us with the necessary training to isolate primary hepatic stellate cells from mice.
Impact This collaboration critically supported our work focusing on the role of macrophages in pancreatic cancer. Specifically, this collaboration contributed to our manuscript describing a novel role of macrophages in pancreatic cancer metastasis (Nature Cell Biology, in revision).
Start Year 2015
 
Description Secretome analysis 
Organisation Korea Research Institute of Bioscience and Biotechnology (KRIBB)
Country Korea, Republic of 
Sector Academic/University 
PI Contribution We have generated several primary cell lines and the secretome thereof. In addition, we have prepared secretome samples from treated or untreated cell cultures. Protein samples were submitted to our collaborators.
Collaborator Contribution Our collaborators have analysed by mass spectrometry global secretomes of our samples.
Impact We have included part of the data in one of our manuscripts which is currently under revision. Secretome data will be made public available after acceptance of our manuscript.
Start Year 2015
 
Description p110g KO mice 
Organisation University of Turin (Università degli Studi di Torino UNITO)
Department Molecular Biotechnology Center (MBC)
Country Italy, Italian Republic 
Sector Academic/University 
PI Contribution Breeding p110g KO mice
Collaborator Contribution Transfer of the p110g KO mice from University of Turin to University of Liverpool
Impact N/A
Start Year 2013
 
Title Cancer Therapeutics 
Description The present invention relates to combinations and pharmaceutical compositions comprising an IGF inhibitor and a chemotherapeutic agent, and their use in treating a 5 proliferative disorder such as cancer (for example a solid cancer such as pancreatic cancer). The invention also provides an IGF inhibitor for use in treating such proliferative disorders in combination with a chemotherapeutic agent, and a chemotherapeutic agent for use in treating such proliferative disorders in combination with an IGF inhibitor and a biomarker for identifying proliferative disorders which have increased responsiveness to 10 the combined treatment. 
IP Reference PCT/GB2016/050092 
Protection Patent application published
Year Protection Granted 2015
Licensed No
Impact We are now applying for funds to perform a clinical trial here in Liverpool to test the novel combinational therapy in PDAC patients.
 
Description Fund raiser event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I have been invested to speak at a fund raiser event organized by a local charity. I have explained in lay terms which are among the biggest challenges in cancer research and how scientist address them. I provided a show case how money provided by research councils and charities is used to fight cancer.
Year(s) Of Engagement Activity 2015
 
Description MRC Science Summer Festival 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact MRC Summer Festival: my team actively participated at the MRC Summer Festival organized by the University of Liverpool. During one week, we provided two lab tours per day to the general public and explained our MRC funded research efforts in lay terms to the visitors.
Year(s) Of Engagement Activity 2016