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Deciphering tumor-promoting mechanisms mediated by neutrophils in vivo

Periodic Reporting for period 1 - CINPinCB (Deciphering tumor-promoting mechanisms mediated by neutrophils in vivo)

Periodo di rendicontazione: 2021-05-01 al 2023-04-30

Immunotherapies are revolutionizing cancer care but are only effective in a minority of patients. Because current treatments selectively target T lymphocytes, manipulating other cell types should create additional therapeutic opportunities. In this project, we focused on a myeloid cell population, the neutrophils, because they can be abundant in the tumor microenvironment, and their presence is often associated with a poor clinical prognosis. Consequently, patients who do not respond to current treatment options may benefit from novel therapeutic strategies targeting these cells. However, only some tumor-associated neutrophils promote tumor growth, and the complexity of these cells is not clearly understood, largely due to the experimental limitations inherent in studies of these cells. Therefore, novel approaches are needed to study tumor-associated neutrophils' mode of action. To address these knowledge gaps and experimental limitations, we began by studying human and murine tumors by single-cell RNA sequencing and histology to reveal the phenotypic complexity of tumor-associated neutrophils, and their conservation in both species. We then developed two experimental approaches to define the role of certain genes in the production of pro-tumor neutrophil responses: 1) we used CRISPR technology to genetically modify neutrophil progenitors that are then adoptively transferred into mice to study their functions in vivo; 2) we used genetic conditional mouse models to manipulate endogenous neutrophils in vivo. With these tools, we have identified several genes that appear to be required in the development of a pro-tumor neutrophil response. These results may have scientific and therapeutic significance, as they not only further our fundamental understanding of the mechanisms regulating neutrophil activities in cancer, but also point to new molecular targets for therapy.
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.
To date, the study of neutrophils has been extremely difficult mainly due to technical shortcomings, and analysis of neutrophils at the single-cell RNA level has been almost impossible. Therefore, novel approaches were needed to study tumor-associated neutrophils’s mode of action. Until the end of this project, we will be able to 1) propose innovative approaches to study tumor-associated neutrophil heterogeneity and mode of action and 2) reveal neutrophil-derived biomarkers and mechanisms of their action.

The data generated for this proposal will be used by the scientific community involved in the study of neutrophil biology in cancer and other disease settings. These technological innovations will be used to uncover mechanisms that regulate neutrophil activities in cancer. Gaining this knowledge is essential because it will deepen our fundamental understanding of immune cell functionality in cancer and will benefit translational work by proposing new prognostic biomarkers and therapeutic targets for clinical use. Furthermore, we expect that the systems proposed here will lay the foundation for research investigating the mechanisms of neutrophil function in any disease setting. All methods and datasets generated in this project will be publicly available for the scientific community and industry to build upon once the work is published.
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