In this application we established a new approach to study neutrophils that not only circumvents prior experimental limitations, but also opens the possibility to uncover neutrophil functions at a mechanistic level in vivo.
We specifically showed that so-called ER-Hoxb8 progenitors can be used to produce unrestricted numbers of bona-fide tumor-associated neutrophils in vivo (Work Package 1;WP1), and that these cells can be gene-edited to study their roles during cancer progression and/or therapeutic intervention (Work Package 2;WP2). In detail,
WP1 - To create an efficient and expandable system for effective mechanistic in vivo studies of neutrophils, we worked on two specific aims: first, we validated the use of bone marrow-derived progenitors that can be ‘immortalized’ to produce unlimited numbers of bona-fide neutrophils in vitro and in vivo. We referred to these cells as conditionally immortalized neutrophil progenitors (CINPs). Second, we 1) proved the ability for CINPs to produce both Siglec-Flow ('bystander') and Siglec-Fhigh (tumor-promoting) neutrophils in vivo in mice bearing KP1.9 lung adenocarcinomas; and 2) established the most optimal conditions for good in vivo engraftment of CINP-derived Siglec-Flow and Siglec-Fhigh neutrophils. This is of particular importance because it allows us to use the CINP-based system for in vivo mechanistic studies.
WP2 - To investigate in vivo mechanisms of pro-tumoral neutrophil activity, we used CRISPR/Cas9, a powerful gene-editing technology, that can be used to knock out genes in mammalian cells. To select genes of interest, we established a new signature-based rescue procedure that can identify neutrophils with low transcript counts from 10x single cell RNA sequencing data. Then, first, we successfully generated gene-edited Cas9+CINPs for all genes of interest. Second, we used the gene-edited Cas9+CINPs for in vivo mechanistic studies.
The results of this project were disseminated and communicated in two international conferences, in online international meetings, as well as in several departmental and interdepartmental meetings and progress reports at AGORA, UNIGE, UNIL and EPFL, which opened up collaborations with other groups in AGORA and outside Switzerland, as well as with industry. In addition, the communication of the project idea to a non-scientific and diverse audience, dominated by children and young students, took place at the European Researcher's Night 2022. In addition, dissemination of the project's data will take place through the publication of an original research article before the end of 2023. To date, this project has not led to the development of intellectual property, so no action has been taken to protect it.
