Naked mole-rat macrophages are easily transformed by c-Myc overexpression

Cancer is one of the leading causes of death worldwide, placing an increasing burden on the population and national healthcare systems and determining a widespread interest in cancer prevention. Naked mole-rats (Heterocephalus glaber) are a highly relevant model for cancer research, because despite their exceptional longevity they show a very low incidence of spontaneous tumors, a hallmark of old age in other species, including humans. Several mechanisms have been proposed as being responsible for protecting naked mole-rat cells from tumorigenic transformation, but to date there is a debate on whether cell intrinsic or cell extrinsic mechanisms are mainly responsible for avoiding tumor formation in this species. This aspect is particularly relevant as it determines how research performed on this animal model could eventually translate into clinical investigations. The main limitations of previous studies can be attributed in part to the limited tools available for research in non-canonical model organisms and in part to the complex models of experimental tumorigenesis in naked mole rats requiring multiple oncogenes or transforming events. In this thesis, I therefore investigated the type of mechanisms responsible for naked mole-rat cancer resistance by developing a simple model of experimental tumorigenesis with overexpression of a single oncogene, c-Myc, in macrophages, since this oncogene is known to be sufficient for transformation of this specific cell type in the mouse. To address the lack of validated protocols for culturing naked mole-rat macrophages, I developed a protocol for obtaining large numbers of naked mole-rat bone marrow-derived macrophages. I could show by RNA-seq and flow cytometry that the cells obtained with this protocol express characteristic macrophages markers. Furthermore, I used the cultured cells to screen for anti-human and anti- mouse antibodies cross-reacting with naked mole-rat cells. With this approach I could identify 13 clones labeling macrophage surface markers that then could be further used to characterize tissue resident macrophage populations, such as Kupffer cells, in the naked mole-rat by spectral flow cytometry. This way I expanded the limited repertoire of protocols and reagents available for this species and established new resources that make it possible to study the naked mole-rat innate immune system with modern tools. Finally, I could show that naked mole-rat macrophages can be transformed by a single oncogene, c-Myc. Myc-transformed cells gained the ability to proliferate long-term in culture and to induce tumor formation when injected in immunocompromised mice. In contrast to previous studies in naked mole rat fibroblasts, requiring more oncogenes and higher expression levels than mouse cells for transformation, I observed no difference of naked mole rat macrophages in their response to c-Myc compared with mouse macrophages. My results clearly show a lack of cell-intrinsic cancer resistance mechanisms in naked mole-rat macrophages, indicating that there is no general barrier to oncogene mediated transformation in naked mole rat cells. This suggests a predominant role of cell-extrinsic mechanisms in preventing tumor formation in naked mole-rats, opening the road to future studies on naked mole-rat tumor protective environment, including their immune system and metabolism.