An autochthonous model of lung cancer in the Naked Mole-Rat (Heterocephalus glaber)

There is growing interest in understanding the mechanisms underlying differences in cancer incidence among species. The naked mole-rat (NMR) is often referenced as “cancer-resistant” and prior studies focused on identifying mechanisms explaining this. However, efforts to assess this in vivo have been limited. Herein, we provide evidence that the NMR presents as a novel autochthonous model of lung tumor initiation, driven by an introduction of the oncogenic Eml4-Alk fusion protein using CRISPR-mediated genome editing. While in mice the inversion alone is sufficient to drive tumorigenesis, the inversion alone was insufficient to drive tumorigenesis in the NMR lung and tumor development required additional losses of the tumor suppressors p53 and pRb. Our findings suggest that the proposed “resistance” of the NMR to the development of cancer may reflect tumor initiation mechanisms that are likely to be comparable to the mechanisms present in human cells. Overall design: We utilized deep sequencing of NMR Rb and p53 in order to detect what mutations were introduced into the NMR lung tissue. To do this, we performed laser capture microscopy on 3 different NMR lungs to obtain specific lung tumor areas as well as non-cancerous regions. We amplified regions surrounding the CRISPR sites and sequenced them using the Illumina Nextera XT kit following the manufacturer?s protocol. Briefly, following the quality control screening of 100ng of each amplicon on a 1% agarose gel, both amplicons for each sample were pooled equimolar and 1ng of input DNA was used for the Nextera tagmentation reaction, followed by PCR amplification and sample indexing. The libraries were purified, quantitated with the Qubit Fluorometer, and screened on the Agilent TapeStation 4200 using the TapeStation High-Sensitivity D1000 Screen Tape . The final libraries were normalized, denatured, and sequenced on the Illumina MiSeq sequencer using a Nano flow cell to generate approximately 20,000 150-base read pairs per sample.