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We inject the tumor cells (from the patient biopsy) into the zebrafish. We do a single cell suspension and then we apply the treatment that the oncologist indicated and we sort out which treatment that specific person responds better to.

Yes

We analyzed the number of disseminated cells in the tail region.

In the 3 days of experiment, we do not have any reaction since the immune system of the embryo is not fully developed yet.

It takes 24 hours and then we can see it.

We can use cell lines or PDX models that are already characterized and use those models with high or low expression of the target protein. Example: if the compound target HER2 we can use two different cells lines, one with high and one with low expression of HER2. Then we would expect that the compound shows anti-tumor efficacy with the cell line with high expression and no effect with the low. Another way to do it is by doing knock-down with cell lines using siRNA.

Compared to rodents: our model requires less amount of your compound; no ethical permission needed; lower cost and faster (months in rodents); more number of animals per group.

Compared to organoids (in vitro):  our model requires less amount of your compound; it is faster (weeks- months in vitro); our model gives the possibility to take into account the tumor microenvironment (not in organoids).

Since January 2023 FDA no longer require animal tests before human drug trials (https://www.science.org/content/article/fda-no-longer-needs-require-animal-tests-human-drug-trials ).

Here is relevant information into FDA approaches:

“FDA’s current regulatory framework permits and encourages the use of new alternative methods to animal testing, as described in regulations and guidance.  FDA supports alternative methods that are backed by science and produce scientifically valid data that are appropriately within a context of use to meet the regulatory need.” “…. What are some examples of alternative methods? … Alternative organisms such as Zebrafish and C. elegans …”  https://www.fda.gov/science-research/advancing-alternative-methods-fda/about-alternative-methods

EMA is in favor of using the 3R principle and this was even before the FDA promoted pharma companies to use it.