Periodic Reporting for period 1 - DC cancer (Development and immunological control of dendritic cell cancer)
Reporting period: 2015-05-01 to 2017-04-30
We generated a new genetically-engineered cancer mouse model that makes use of the recent finding by the host lab that the Clec9a gene is expressed specifically within cells of the DC lineage. We used this model to study the aetiology of DC cancer development. DC cancer was observed with 100% penetrance in Clec9a Kras-G12D mice, which all succumbed to neoplastic transformation within the DC lineage. Flow cytometric analyses over time established that high accumulation of transformed DCs occurred in multiple lymphoid and non-lymphoid organs and was evident early in life, from week 4 after birth. DC cancer formation showed 100% penetrance in mice and we did only observe minor heterogeneity between individual mice. However, further analyses revealed that cancer formation is dominated by the CD8a+ subset of DCs, which accumulated over time, indicating that this subset has a stronger potential to form tumors in Clec9a Kras-G12D mice.
We additionally analysed whether oncogenic transformation of DCs affects their immunological phenotype and function. Cancer DCs did not show signs of activation but could be activated by stimulation, evident by the upregulation of costimulatory markers and cytokine production. Furthermore, in vitro culture assays with T cells established that cancer DCs were capable to induce T cell activation and stimulation, suggesting that DCs in Clec9a Kras-G12D mice retained a normal phenotype and functional ability despite their oncogenic transformation. Furthermore, we did not detect changes in the immune cell repertoire in Clec9a Kras-G12D mice, suggesting that cancer DCs are immunologically invisible and ignored by the immune system. This was supported by the finding that crossing Clec9a Kras-G12D mice to Rag1-/- mice did not show accelerated DC cancer development, despite T cell deficiency. However, adoptive transfer of cancer DCs into immune competent mice resulted in immune rejection, an important finding with implications for DC cancer therapy. We complemented our analyses of T cells by analysing further potential immunological barriers that could hinder DC tumor development. To this end, we generated bone marrow chimeric mice in which neoplastic transformation is restricted to a very low frequency of transformed DC. In these experiments, we discovered that a small number of transformed DC precursors is sufficient to evoke rapid DC cancer development and DC cancer development can occur in the presence of untransformed DC.
Taken together, our data provide an in-depth characterisation and fundamental insights into the biology of DC cancer development. Our experiments establish that DC cancer formation is not controlled by immunological barriers in mice, and DC cancer cells seem to be ignored by the immune system. These findings have important implications for the design of targeted immunotherapy against DC tumors.
Dissemination of results
Most of the results of this project have been published in a peer-reviewed manuscript in the Journal of Immunology (See uploaded publication).
Furthermore, results from this project have been disseminated to the scientific community by several oral presentations at national and international meetings.
This project allowed us to generate several DC cancer cell lines, which are available as a biological tool to the scientific community and already used by the several labs at the Francis Crick Institute.
Furthermore, Clec9a Kras-G12D mice are available as a tool to study the formation of DC cancer. In addition to this and beyond the original proposal, these mice have been crossed to Batf3-/- mice, allowing to study neoplastic transformation of CD11b+ DCs; and Rag1-/-Clec9a Kras-G12D mice to study immune-editing of DC cancer in future projects.
Secondly, my postdoctoral work on DCs and cancer allowed me to start a follow up project in which I am investigating the mechanisms that cancers use to impair DCs within tumours. This project was recently submitted for publication.
Thirdly and most importantly, the funding of my postdoctoral work in Caetano Reis e Sousa’s lab allowed me to develop my own area of research interest within the field of cancer immunology. The unique combination of expertise, tools and techniques I acquired during my research as a Marie Sklodowska-Curie fellow ideally positioned me to reach the next level of professional maturity and I successfully acquired grant money for a 5-year junior research group at the TU Munich, Germany, starting next year.