Dottorato in SCIENZE DELLA VITA

Titolo della tesi: Caenorhabditis elegans as a biosensor for the early detection of cancer metabolites in urine samples

The discovery of cheap and non-invasive diagnostic strategies for the early detection of cancer is an urgent priority since treatments are more effective if applied early in the course of the disease. Cancer was shown to deeply alter metabolism opening the door to the development of a wide range of diagnostic tools based on the detection of cancer metabolites as early biomarkers in biological fluids. Thanks to their extraordinary sense of smell, dogs and mice were shown to be able to detect small molecular weight organic compounds as cancer biomarkers in urine samples. However, the introduction of mammals into the clinical practice is not a trivial issue for both experimental and ethical concerns. Conversely, small invertebrate organisms may represent a suitable option for the development of new technologies to promptly diagnose solid tumors. Recently, it has been shown that the nematode Caenorhabditis elegans (C. elegans hereafter) displays attraction towards urine samples collected from patients affected by a wide variety of tumors but avoids those from healthy subjects. Aim of my PhD project was to provide a proof of concept for the development of a microfluidic lab-on-chip platform for the early detection of cancer metabolites in women with breast cancer (BC) by using C. elegans as a biosensor. The nematode has a small nervous system composed of only 302 neurons with a completely mapped connectome and a highly developed chemosensory system that enables it to detect a wide variety of volatile and water-soluble cues. Chemotaxis assays confirmed and extended previous data highlighting the ability of the nematode to discriminate between urine samples collected from BC women prior to chemotherapy and healthy subjects, with a sensitivity (i.e., true positive rate) of 78% and a specificity (i.e., true negative rate) of 97%. Of note, the response was shown to be strongly influenced by hormones in fertile women. Specifically, we observed a positive correlation between the chemotaxis index and the FSH, LH and estradiol release preceding ovulation, and the progesterone peak during the luteal phase, indicating the presence of two relatively narrow time windows for sample collection in young women. Behavioral assays performed on animals in which the AWC sensory neurons were genetically ablated demonstrated an essential role of these neurons in sensing cancer odorants. Furthermore, chemotaxis experiments performed on mutant animals knock-out for genes encoding G-protein coupled receptors (GPCRs) expressed in AWC neurons allowed us to identify candidate receptors which are likely to be involved in binding cancer metabolites. CRISPR-Cas9 engineered C. elegans lines have also been generated to assess the contribution of additional AWCs-expressed GPCRs for which knock-out strains were not available to the attractive response towards cancer samples. Chemotactic data were then mirrored by calcium imaging analyses. Finally, gas chromatography/mass spectrometry (GC-MS) analysis identified a number of metabolites showing significant differences in their relative abundances between cancer and control samples, providing a panel of putative molecules responsible for the attractive behavior of the nematode. We finally established the C. elegans preference towards these compounds and found that the nematode shows chemotactic responses to a subset of them. Dissecting the biochemical mechanisms mediating the ability of the nematode to “recognize” cancer metabolites is expected to generate new insights on the identification of relevant biomarkers for the early diagnosis of cancer, cancer metastasis and relapses, one of the major public health challenges of the 21st century.