State-of-the-art mouse models as an innovative preclinical tool to study the current challenges in mammary gland inflammation

The past decade, the laboratory of Biochemistry, based at the Faculty of Veterinary Medicine (Ghent University, Belgium), has gained unique expertise in the development and validation of preclinical in vivo models to study mammary gland inflammation. Under the supervision of Prof. dr. Evelyne Meyer, state-of-the-art mouse models have been developed and validated as tools to gain insight in the most prevalent diseases that impact mammary gland health: mastitis and tumour formation. Mammary gland infection or mastitis most frequently occurs in dairy cows and other ruminants, whilst mammary tumour formation is more relevant in humans, dogs and cats.

Mammary gland infection and tumour formation are associated with inflammation

Bovine mastitis occurs when micro-organisms invade the cow’s udder. Although preventive and curative use of antibiotics combined with good hygiene practices helps to control this infection, it remains impossible to fully eradicate bovine mastitis. Upon infection of the mammary gland, the cow’s immune system is triggered resulting in inflammation and disease. With growing concern regarding the ecological footprint of dairy products and the overuse of antibiotics in the sector today, the currently used intramammary preparations used in the treatment of bovine mastitis are being questioned. Most of these preparations are also given preventively as dry tow therapy and contain antibiotics that are considered critical in human health care. Therefore, the dairy industry is moving towards a more sustainable and eco-friendly way of milk production. Our mastitis mouse models have been adapted with state-of-the-art techniques and technical know-how to screen novel candidate therapy compounds that are under development to treat mammary gland infections and its associated inflammation.

Mammary tumours are the main cause of cancer in women today and frequently affect dogs and to a lesser extent, cats.  Although innovative therapies such as immunotherapy are emerging in veterinary medicine, they frequently clash with the financial possibilities of pet owners. In the attempt to more effectively treat aggressive mammary tumours, the role of the inflammatory tumour micro-environment has more recently been elucidated in mice. The Meyer-group established and fully-characterized two complementary immunocompetent mouse models that allow screening of new compounds impacting this inflammatory micro-environment. However, future preclinical research with a relevant intraductal mouse model in veterinary oncology remains necessary.

Immunocompetent mouse models to study mammary gland inflammatory disease.

Mice are considered a valid model for other mammalian species, including the cow, due to multiple anatomical and functional similarities. Mice are also easy to house, cheap in maintenance and have a short reproduction cycle. The latter is especially important since mice are required to lactate when mastitis or mammary tumours are experimentally induced. Whilst mastitis is typically established in the fourth mammary gland pair as a model for cows, the third pair is preferred to induce breast cancer (Figure 1A). The fourth mammary gland pair is located in the inguinal region of mice, contains mammary lymph nodes and therefore anatomically resembles the cow’s udder. Upon infection, mastitis-derived bacteria are grown, washed and serially diluted until the desired concentration. Depending on the mastitis pathogen, the amount of bacteria inoculated is adapted i.e. one hundred microliter containing 102 colony forming units (CFU) is preferred for Staphylococcus aureus (S. aureus) (Figure 1B), whilst 103 CFU is needed to establish mastitis with Streptococcus uberis (S. uberis). Apart from the latter two mentioned pathogens, our group also has in-house expertise with Escherichia coli (E. coli) and coagulase-negative Staphylococci. During the inoculation procedure, mice are always under general gas-anaesthesia using isoflurane and given post-chirurgical pain medication. While they develop disease, they are closely monitored by experienced animal caretakers and a veterinarian, using internationally accepted clinical scoring schemes. Our bovine mastitis experiments never exceed 48 hours. All our procedures are approved by an independent ethical committee and the animals are housed in the best possible conditions concerning their well-being. Moreover, mice are only bred on purpose and therefore, we prefer a stock of frozen embryos or purchase over continuously keeping animals alive. This also counteracts genetic drift and therefore ensures the genetic stability of our population.

Our group combines the expertise of various mice strains and adapts the choice of the animals to the goals in mind. Outbred (CD-1 strain) mice are generally bigger and therefore ideal for bovine mastitis experiments. In contrast, inbred (BALB/c and C57BL/6 strains) mice are preferred for mammary tumor experiments, since the mammary tumour cells that are used in these strains closely mimic the disease. In addition, the Meyer-group also has a genetically modified C57BL/6 mouse strain available that has a luciferase gene knocked into the NF-κB gene. These mice can painlessly be injected with luciferine and subsequently make inflammation visible through emission of light (Figure 1C). So yes, we actually make the mammary glands of mice glow like a firefly! In fact, we also have the ability to label tumour cells or bacteria this way. Due to this emission of light, our group can closely follow the diseased mice over time without a need to kill them. This innovative kinetic monitoring strategy therefore perfectly fits within our reduction strategy to minimise the amount of mice needed. Once the experiment has been finished, our mice are humanely euthanized. Mammary glands, and if necessary: blood, lungs, liver, spleen and lymph nodes are harvested. Thereafter,  our in-house expertise applies the latest technology for a complete and in-depth analysis of these samples. These include histology, immunohistochemistry, ELISA, multiplex assays and our core expertise, flowcytometry. The combination of these state-of-the-art molecular technologies makes a complete characterization, up to individual cell level and intercell communication, possible.

The results we gain out of our innovative mouse models today, pinpoint towards a solution for mammary gland inflammatory disease tomorrow.

Since the Meyer-group owns more than ten years of core expertise within the subject matter, several publications in international peer-reviewed journals and PhD theses have been made. The mastitis mouse model successfully validated new compounds as an alternative or add-on strategy to the currently used intramammary preparations. These include i.e. probiotics, plant-derived compounds and last but not least bacteriophages. Our mastitis mouse model also contributed to the potential of hamamelitannines, antibacterial molecules derived from the witch hazel tree, as an add-on strategy to the classic antibiotics (i.e. cefalexin) used in the treatment of S. aureus bovine mastitis. More recently, a collaboration with EpiBiome evaluated the potential of a bacteriophage cocktail as a novel mastitis strategy. Bacteriophages are viruses that infect and subsequently lyse bacteria, while at the same time amplifying themselves using the host bacterium. Our preclinical model showed that this cocktail significantly improved S. aureus mastitis and decreased bacterial counts. Last year, a PhD grant by the research foundation of Flanders was offered to further develop a promising bacteriophage-derived enzyme as alternative antimicrobial for Gram-positive bovine mastitis. This project is currently ongoing and runs in close cooperation with the University of Maryland (USA) and both faculties of bioscience engineering at KULeuven and Ghent University. Furthermore, the preclinical mastitis mouse model also contributed to more fundamental insights in mammary gland infection. In example, the role of two bacterial virulence genes (EntA, SigS) have been found eligible as a potential target in the treatment of bovine mastitis. A European Animal Health and Welfare collaboration (ANIHWA) in which our preclinical mouse model was involved, further elucidated the role of glycoprotein chitinase 3-like 1 (CHI3L1) in the pathogenesis of E. coli mastitis. On the other hand, the breast cancer mouse model supplementary clarified the role of polarized macrophages in this disease. Our model has now successfully been deployed as screening tool for a novel, anti-tumour c-MET inhibitor developed by an oncology company as an add-on strategy to the currently used cisplatin chemotherapy. In the near future, more studies will be performed together with the pharma industry to validate novel anti-cancer therapies in our breast cancer mouse models.

In conclusion, mice are considered a valid preclinical tool to translate in vitro findings (i.e. research conducted with bovine mammary cells) towards the target species (i.e. the cow). This first preclinical onset remains indispensable in the development of novel compounds targeting mammary gland disease and inflammation. Therefore, the Meyer-group wants to offer valid and high-quality mouse models combined with state-of-the art analytical techniques to address the challenges new compounds are facing when transitioning from in vitro studies towards clinical trials.

Text and illustrations: Niels Vander Elst and Evelyne Meyer