The Rutkowski Lab in the Department of Microbiology, Immunology and Cancer Biology recently discovered that an unhealthy gut microbiome is linked to increased metastasis — the spread of — breast cancer in mice. The microbiome is the collection of microorganisms that live both on and within humans, as well as other animals. Principal investigator Dr. Melanie Rutkowski explained that patients with unhealthy microbiomes have worse outcomes when diagnosed with breast cancer. This led the team to think about the microbiome’s potential role in influencing cancer progression. “These microorganisms have a fundamental role in influencing a lot of host physiological functions but also the immune system in general, which is sort of why we're interested in it,” Rutkowski said. Rutkowski explained that microbiome health is largely determined by biodiversity. “When [the microbiome] is less diverse, it's considered more inflammatory and less healthy,” Rutkowski said. To explore their hypothesized connection between the microbiome and cancer outcome, the team first used antibiotics to artificially create an unhealthy microbiome in mice. The antibiotics used were non-absorbable, meaning that they only target the gut microbiome. Mice were either treated with a cocktail of several antibiotics in the experimental group or water in the control group for two weeks before their resting period of four days. Afterwards, the mice were injected with breast cancer tumor cells, and tumor growth was observed. Lab specialist Francesca Azar, whose primary role in the research involved removing and processing the mice tissue, explained some of the work done to analyze the tumor dissemination after antibiotic administration. The tissues are digested using enzymes so that single cells can be separated out. Then, the researchers can use techniques such as staining to prepare the tissue for methods of analysis such as flow cytometry, which allows them to identify the types of cells present. “[Removing and processing tissues from the mice] involves enzymatically mechanically digesting the tissues and creating these single-cell suspensions,” Azar wrote in an email to The Cavalier Daily. “From that point, downstream techniques such as staining for flow cytometry analysis happens,” she added. Claire Rosean, a postdoctoral researcher and first author on the published paper about the research, explained the major differences between the antibiotic-treated and control mice. “We found that mice that had been pretreated with antibiotics had enhanced tumor cell dissemination to lymph nodes, lungs, and peripheral blood at both early and advanced timepoints after tumor initiation,” Rosean wrote in an email. In other words, the cancer cells injected into antibiotic-treated mice were more successful in spreading to areas typically associated with metastasis. Rosean also explained that the researchers found more macrophages — a type of cell used by the immune system to remove debris and foreign particles — as well as more collagen depositions in mice treated with antibiotics. “There have been studies that suggest that a more collagen-rich environment can set up a positive niche for tumor cells to set up an environment there and proliferate,” Rosean said regarding the team’s observation of increased protein deposits. Both Rosean and Rutkowski clarified that their study focused on cancer metastasis rather than initial development and growth. “We're not saying anything about the risk of getting cancer or how the primary tumor grows … but more in terms of how it affects metastasis, which we know [is] basically the major factor which will determine that patient's prognosis with breast cancer,” Rosean said. Rosean explained that the team was able to confirm that some aspects of the altered microbiome itself was responsible for the increased spread of the breast tumor. They used a technique known as fecal microbiota transplantation, in which the feces of certain mice are implanted into a host mouse. “If you took what I'll call an unhealthy microbiome from a mouse and transplanted it into an otherwise healthy mouse, we were able to make that breast cancer more disseminated once we initiated cancer in those animals,” Rosean said. The researchers acknowledged that much more work must be done to confirm whether the phenomenon of the microbiome affecting cancer spreading also occurs in humans. Rutkowski discussed the lab’s next steps with their findings. She explained that even though microbiomes vary greatly across individuals, the metabolites, or chemical products, produced by the microorganisms are relatively conserved and hence make for good pharmacological targets. “I think targeting those products would be a more feasible way to develop a more amenable therapy that could be utilized across multiple individuals and possibly multiple cancers,” Rutkowski said. Targeting these conserved molecules may be more feasible because, according to Rutkowski, changing one’s microbiome is complicated. “I think it's really hard to change the microbiome of someone if they don't also change their lifestyle factors,” she said. She continued to say that maintaining healthy lifestyle habits such as proper diet, sleep and exercise can help maintain a healthy microbiome. Azar discussed the finding’s implication that cancer treatment should be approached with a broader perspective. “I think that this finding lends itself to the idea that we do not need to approach cancer treatment from a single avenue, and instead we can aim to take a more holistic approach to its treatment,” Azar said. She also mentioned the possibility of targeting specific strains of bacteria, in contrast to Rutkowski’s metabolite-focused approach. “If we implicate specific populations of bacteria in the progression or spread of cancer, they may be useful targets for future treatments,” Azar added. Rutkowski said the team hopes to soon collaborate with clinicians to study the correlation between microbiome health and cancer progression in recently-diagnosed patients. “[It would require] longitudinal sampling of their [patients’] microbiomes to see whether, if they have an unhealthy microbiome prior to a breast cancer diagnosis, is that truly predictive for whether or not they're going to develop metastatic disease years to decades later,” Rutkowski said.