Anthony Caruso and Faculty Mentor: John Chaston, Plant and Wildlife Sciences

Introduction
It has been established by past scientific research that the gastrointestinal microbiome plays a
defined role in many human diseases. Some of these diseases include inflammatory bowel
disease, autism, hypertension, and even cancer. A common symptom seen in many of these
diseases is the dysbiosis of intestinal microbiota. Studies investigating several of these ailments
have shown that model organisms can be rescued from negative symptoms through the
addition of health promoting bacteria to their gastrointestinal tracts. While these fascinating
correlations between levels of gut microbiota and disease have been well established, the
specific genes of the bacteria and the inter-organism chemical pathways that participate in this
process are widely unknown. This is partly due to the difficulty in using metagenomic methods
since the specific micro-populations differ vastly from one organism to another. Drosophila
melanogaster provides a realistic opportunity to explore the effects of bacteria genotypes on
host phenotype because of the ease of controlling the environment and microbiome or the flies
in laboratory conditions.
Methodology
By crossing flies with mutations for the Ras oncogene and the Scrib gene respectively, we were
able to create a generation with unregulated cellular growth and metastatic behavior in the eye
disk cells of the larvae. This combination of mutations is lethal before pupation so all
observations were made in the larval stage. The tumor development and metastasis was
monitored by means of GFP-labeled cells in the eye-antennal discs. A high resolution
microscope with the ability to focus UV light was used to image the cancer metastasis. The eggs
resulting from the above mentioned cross were collected after 20 hours of the two parental
strains being in the same container together. The eggs were then made axenic through a wash
in a solution of bleach and purified water. After this, the axenic eggs were placed on a sterile
food medium of glucose, yeast, and agar in 16 vials. Half of these vials were left axenic, while
the others had five strains of bacteria introduced. These five strains of bacteria are good
representatives of the most common strains found in the intestines of Drosophila. The axenic
eggs would develop into larvae completely devoid of any bacteria in their gastrointestinal tract.
The others would develop into larvae with only the five strains introduced living in their
intestines. We hoped to discover significant differences in the levels of metastasis between the
axenic and gnotobiotic flies. We would then run a metagenome-wide association study on the
microbial DNA of the strains introduced to identify cancer metastasis associated markers.
Results
We were able to successfully confirm that crossing flies with Ras and Scrib mutations,
respectively, resulted in lethal metastatic cancer of the eye disc cells. This was done by allowing
eggs from the cross to hatch into larvae without undergoing the sterilization process to create a
control group. This yielded plentiful larvae which all exhibited observable cancer metastasis.
However, we were unable to detect any significant differences in metastasis between axenic
larvae and larvae that had been exposed to the five strains of bacteria. This was because both
groups yielded a few unusable larvae or none at all. Three attempts were made to produce
axenic and gnotobiotic larvae, all of which failed to produce usable samples. We investigated
the methods used to generate the axenic and gnotobiotic groups on a control group of flies with
no mutations. On two separate occasions these methods succeeded in producing healthy larvae
in unmutated fly strains. Because we were able to create healthy mutant larvae when the eggs
were not sterilized as well as the fact that unmutated eggs were able to be sterilized while still
developing normally, we concluded that mutations to the Ras and Scrib genes must cause a
change in the ability of the eggs to withstand sterilization.
Discussion
While confounding variables prevented our experiment from producing the desired experimental
groups of flies, we maintain that Drosophila melanogaster is still a viable model to investigate
the microbiome’s influence on cancer metastasis. Multiple genetic mutations in Drosophila have
been demonstrated to cause the development of cancerous cell masses and many of these
mutation are also homologous with mutations that cause cancer in humans. In addition to this,
fast generation time and the easiness of maintaining them in a laboratory make Drosophila an
excellent model organism for investigating cancer. We plan to continue investigating the
interaction between the gut microbiome and cancer metastasis in Drosophila by adapting our
experimental procedures. We have identified two different changes we might make to our
methods in order to produce usable larvae. We hypothesize that the cancer causing mutations
are also causing the protective walls of the eggs to become weak and unable to survive the
sterilization process. One way that we might circumvent this problem is to simply lower the
concentration of bleach in the sterilizing solution. If this proves to be unsuccessful, the second
change we can make is to raise up two sterile groups of flies, each containing either the Ras or
Scrib mutation. We would then allow normal mating, but cause this to also take place in a sterile
environment. While this approach would take much longer and would be much more tedious, it
should eliminate the need for the sterilization process on the eggs resulting from the cross.
Conclusion
The correlations between changes in the gut microbiome and disease are too well established
to ignore. A large variety of diseases cause consistent changes in the levels of gut bacteria of
many organisms. Drosophila melanogaster provides a unique opportunity to test hypotheses
with specific microbiota on a large scale with the potential to shed light on the metastatic
processes in humans and open the way for additional treatment options.