Targeting the immune microenvironment in pancreatic cancer

Lapienyte, Laura (2021) Targeting the immune microenvironment in pancreatic cancer. PhD thesis, University of Glasgow.

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Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death worldwide. Despite the improved worldwide living standards and increased access to healthcare, pancreatic cancer incidence has increased over the past few decades. Regardless of the impressive advances in the field of cancer therapeutics, surgery remains the only potentially curative treatment for pancreatic cancer. However, most of the patients present with an advanced stage of pancreatic cancer at the time of diagnosis, for which there are very few available therapies.

Pancreatic cancer is characterized by a high degree of tumour heterogeneity, with the tumour microenvironment, also known as stroma, accounting for 80% of the total tumour volume. Pancreatic tumour stroma is one of the main hallmarks of pancreatic cancer and is characterized by extensive deposition of extracellular matrix (ECM) components, abundance of proliferating cancer-associated fibroblasts (CAFs), a significant myeloid cell compartment, but a distinct exclusion of T cells. Although deposition of desmoplastic tumour stroma was originally considered as a bystander in carcinogenesis, recent studies have highlighted its role during tumour progression and in facilitating therapeutic resistance.

Improved understanding of the immune system and its role in cancer development and progression has led to impressive advances in the field of cancer immunotherapy over the last decade. However, the success of immunotherapy has not translated to the treatment of pancreatic cancer. Tumour associated macrophages (TAMs) are recognized as critical drivers of immune escape in the tumour microenvironment. Thus, strategies that can abrogate this effect serve as an attractive option for cancer therapeutics. Our group has previously demonstrated that macrophage targeting, via CSF1 receptor inhibition, significantly increased survival in pancreatic tumour-bearing Pdx1-Cre, LSL-KrasG12D/+, LSL-Trp53R172H/+ (KPC) mice.

I sought to investigate the role of macrophages in the pancreatic cancer microenvironment. I hypothesised that macrophage infiltration to the pancreas would be dependent on the chemotactic signalling through the C-C motif chemokine receptors (CCRs). Thus, I generated KPC mice lacking chemokine receptors CCR1, 2, 3 and 5. By using Pdx1-Cre, LSL-KrasG12D/+, LSL-Trp53R172H/+. CCR1-5-/- (KPC CCR1-5-/-) mice I aimed to establish the role of infiltrating macrophages in tumour initiation, development, and progression.

Through a series of in vivo experiments I characterized KPC CCR1-5-/- mice, demonstrating the absence of CCR2 in primary tumour tissue as well as significantly reduced levels of Ly6Chi inflammatory monocytes in peripheral blood. Assessment of tumour initiation in KPC and KPC CCR1-5-/- mice at 6 weeks of age revealed no significant changes in the number or grade of early precursor lesions, also known as pancreatic intraepithelial neoplasias (PanINs), between the cohorts. Most importantly, an aging experiment demonstrated that KPC mice lacking CCR1,2,3 and 5 have no survival benefit when compared with KPC mice. Extensive studies of the tumour microenvironment showed no indication of reduced desmoplasia in tumours from KPC CCR1-5-/- mice. Moreover, immunohistochemical analysis of end point tumours revealed high macrophage abundance in KPC CCR1-5-/- mice. This indicated that tissue resident macrophages are sufficient to sustain tumour growth and maintain the fibrotic tumour microenvironment observed in KPC mice. Further studies using pharmacological approaches to inhibit macrophages from an early timepoint in KPC and KPC CCR1-5-/- mice revealed significantly reduced survival in both genotypes compared with the untreated controls of the same genotypes. These data suggest that during the early stages of tumourigenesis, macrophages may play a tumour suppressive role.

Our previous study of CSF1R inhibition in tumour-bearing KPC mice, revealed an upregulation of molecules associated with immune activation. What is more, we observed an increase in infiltration of CD19+ B cells in the treated tumours. Together, these data indicated an increase in local adaptive immunity. In contrast, recently published papers focusing on the role of B cells in pancreatic cancer reported a pro‐tumourigenic role of B cells. It was shown that transplanted pancreatic ductal epithelial cells exhibit reduced growth in B cell deficient mice when compared with wild‐type mice. Due to the conflicting data, I aimed to assess the effect of B cells on pancreatic cancer in a more clinically relevant model. I generated KPC mice deficient in mature B cells, by crossing Pdx1-Cre LSL-KrasG12D/+, LSL-Trp53R172H/+ mice with Ighm-/- mice. This B cell deficient KPC mouse model was characterized using flow cytometry and immunohistochemical approaches to confirm the absence of B cells. Interestingly, survival analysis revealed that the lack of B cells had no effect on tumourigenesis. The immune profiling of the end point tumours revealed no apparent T cell defects.

To rule out the possibility that any tumour-suppressive effects of B cell depletion in established tumours could be masked by tumour-promoting effects of B cell depletion during tumour initiation, I used a pharmacological approach to deplete B cells in mice with established tumours. Mice with confirmed tumours were treated with the anti-CD20 antibody. Analysis of both tumour growth, as well as survival data, demonstrated no evidence of advantageous effects of B cell depletion. Finally, I wanted to further address the differences observed in the role of B cells seen between the published articles and the data I generated. Therefore, I aimed to investigate the effect of B cells in syngeneic transplant experiments in the B cell‐deficient mice using KC and KPC primary cell lines. I observed that mice implanted with KC cells survived consistently longer than mice implanted with KPC cells. This observation was made for WT and Ighm-/- mice. However, I did not observe any survival differences between WT and Ighm-/- mice transplanted with either KC or KPC tumour cells. Overall, this indicates that B cells do not play a significant role in tumour development in syngeneic allograft models, at least in our hands. This further supports the results observed in the B cell deficient genetically engineered mouse model (GEMM) of pancreatic cancer.

Overall, my data, alongside previously published studies, suggest that depletion of certain immune cell subtypes can elicit opposing effects. Data in this thesis provide further evidence that the selection of study models often lead to discrepancies in the studies of tumour immune microenvironment. The data I present imply that targeting specific signals that promote tumourigenesis rather than specific cell populations might be more beneficial in fighting tumourigenesis. Further studies are needed to allow the development of efficient immune-therapeutic approaches for pancreatic cancer.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Supported by funding from Pancreatic Cancer UK.
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
Supervisor's Name: Morton, Professor Jen
Date of Award: 2021
Depositing User: Theses Team
Unique ID: glathesis:2021-82628
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jan 2022 15:20
Last Modified: 08 Apr 2022 16:59
Thesis DOI: 10.5525/gla.thesis.82628
URI: https://theses.gla.ac.uk/id/eprint/82628

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