Dissertação de Mestrado Investigação Biomédica 2023 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

Immunotherapy, particularly immune checkpoint blockers (ICB), constitute an important breakthrough in the treatment of cancer. Nevertheless, the success of these therapies is quite limited, possibly due to additional innate immune-suppressive mechanisms present in the tumour microenvironment (TME). To increase response rates, it is critical to discover new molecules that can revert this immune-suppression and fully activate the immune system to fight cancer. During the development of the zebrafish xenograft model for drug testing and personalized medicine, my host lab discovered that different human cancer cell lines exhibit distinct clearance profiles. Indeed, they found that while some cancer cells can evade the host innate immunity and are barely eliminated (“progressors”), others are efficiently cleared by the host in just four days (“regressors”). This discovery opened the opportunity to screen a 774-compound Food and Drug Administration (FDA)- approved Drug Library in “progressor” zebrafish xenograft models, to identify compounds capable of enhancing innate-tumour clearance. Ultimately, the idea is to combine these compounds with ICB, thus harnessing both arms of immunity against cancer and possibly increasing the response rates of current immunotherapies. This last step will imply the use of mice with a functional adaptive immune system, and for that purpose, cancer mouse models, such as MC38 colorectal cancer (CRC), E0771 breast cancer (BC), and B16-F10 melanoma, will be employed. These models have been extensively used to find molecules to sensitize tumours to ICB, as they only respond to ICBs when combined with compounds that increase immunogenicity or convert the immune-suppressive TME into a permissive one. My host lab has already screened more than half of the library, with 560 compounds tested in human CRC (resulting in 14 validated hits) and 443 in human breast cancer (15 validated hits). In order to speed up the drug selection process and minimize unnecessary costs and animals in future pre-clinical mice studies, my work was focused on re-screening the top hit compounds in zebrafish injected with cancer cell lines derived from the aforementioned mouse models. I started by generating and characterizing MC38_CRC, E0771_BC and B16-F10_melanoma zebrafish xenograft models, and results suggest that all of them are “progressors”, although they present distinct clearance profiles and induce different immune microenvironments in the host. Also, MC38_CRC and E0771_BC revealed to be sensitive to the three tested drugs (Scopolamine hydrobromide, Tiotropium Bromide, and Methyldopa Sesquihydrate), whose effect was shown to be dependent on macrophages. While Scopolamine hydrobromide induced a slight increase in macrophage and neutrophil infiltration in the TME; the treatment with Tiotropium Bromide resulted in an increase in pro-inflammatory and phagocytic macrophages, and its combination with Pembrolizumab immunotherapy boosted tumour response to the treatment. Collectively, my results highlight the potential of zebrafish xenograft model as an in vivo platform for drug screenings, particularly, for identifying innate immunomodulators that can boost innate-tumour clearance, and possibly complement adaptive immunotherapies in the future