Using oncolytic viral therapy to target the tumour microenvironment in chromosomally unstable cancers
The cancer treatment landscape has been transformed by the success of immunotherapies over the past decade. However, most patients still receive no benefit from this approach, due to tumour-mediated immunosuppressive mechanisms. One of these immunosuppressive strategies involves cancer-associated fibroblasts – these are otherwise normal stromal cells which have been hijacked by cancer cells rewiring pathways within these fibroblasts to promote tumour growth and immunosuppression. Previously, the Parkes group has identified that intrinsic characteristics of the cancer cells, e.g. chromosomal instability, directly impact on the behaviour of fibroblasts in the tumour microenvironment.
Commercial partner Theolytics have identified and developed novel oncolytic viral therapeutics shortly entering the clinical setting. However, the behaviour of these therapeutic agents in a fibroblast-rich, immunosuppressed tumour microenvironment is not currently known and of key interest as these are typically cancers resistant to other immunotherapeutic approaches. Therefore, in this project, using their lead therapeutic candidates, 2D and 3D co-culture models will be used to characterise the relationship between tumour cell characteristics, fibroblast phenotype and response to oncolytic viral therapy.
(1) Investigate the interaction between cancer cells and fibroblasts in response to oncolytic viral therapy. In order to assess the direct effect of co-culture of cancer cells and fibroblasts measuring response to oncolytic virus, novel isogenic cancer cell lines of increasing chromosomal instability (produced from the Parkes lab) will be cultured with immortalised cancer-associated fibroblast lines derived from patient samples. Fluorescently-labelled cancer cells and fibroblasts will be disaggregated for RNA and chemokine profiling in response to virus. Fibroblast phenotype will be further assessed using flow cytometry for expression of cancer associated fibroblast marker expression.
(2) Characterise the role of matrix organisation and stiffness in response to oncolytic viral therapy. Fibroblasts are the principal producers of matrix proteins in the tumour microenvironment, which subsequently affects the mechanical properties of the tumour including stiffness and the ability of cells to migrate through the tumour. The effect of matrix stiffness on oncolytic virus response will be assessed using artificial matrices of increasing stiffness. In these matrices, organoids (representing low and high-chromosomal instability) will be co-cultured with fibroblasts. The ability of oncolytic viruses to induce cell death in the context of increasing stiffness, relevant to the tumour microenvironment, will be measured. The activity of oncolytic virus to infect cells and replicate will be assessed in the context of matrix stiffness.
(3) Investigate the effect of fibroblast activation and matrix stiffness on immune cell activation. T cells cultured in conditioned media from cancer cell-fibroblast co-cultures will be analysed for ability to activate, proliferate and recognise tumour antigen. Using models in aim (2), 3D co-cultures of organoids, fibroblasts and immune cells (derived from blood samples) will be used to characterise immune cell activation, proliferation and cytotoxic activity in response to oncolytic viral therapy.
This project will characterise a novel therapeutic agent using newly developed cell lines and fibroblast co-culture, as well as comprehensive near-patient 3D-models of the tumour microenvironment.
The successful applicant will receive extensive training in 3D culturing of organoid models, working with fresh tissue and establishing novel cell lines. Candidates will be trained in cell sorting, microscopy and RNAseq analysis. The Parkes and Buczacki labs are established teams including postdoctoral fellows, DPhil students and technical support. The techniques described in this proposal are familiar to staff and students in the Parkes and Buczacki labs. The student will be strongly encouraged to attend the two week R for data science and genomics course (University of Oxford) to enable analysis and data visualisation of the generated RNAseq data within this project. From Theolytics, extensive expertise in oncolytic virus techniques is provided. This includes access to novel oncolytic viruses, training in handling of viruses and relevant culture techniques. As the Parkes and Buczacki labs are housed within Oncology and the new Centre for Immuno-Oncology, all suitable equipment for detailed analysis is available, including the Cytek Aurora for flow cytometry analysis. The Parkes lab has expertise in co-culture and has already generated the chromosomally unstable 2D lines and novel fibroblast cultures described within this proposal. The Buczacki lab has extensive expertise in organoid generation and culture, including fibroblast-organoid co-culture. Within oncology and NDS, students will be immersed in regular attendance at internal and external seminars, and strongly encouraged to engage with a wide multi-disciplinary team to enable them to lead and shape the project with their own ideas.