Phd in ADVANCED SURGICAL TECHNOLOGIES

Thesis title: Le cellule tumorali staminali quiescenti chemioresistenti. Basi molecolare della resistenza nel tumore del colon retto

ABSTRACT Despite the advancements in standard cancer treatments, the recurrence rate in patients affected by colon-rectal cancer (CRC) remains high, often due to the development of chemo-resistance to conventional therapy. Unfortunately, our ability to finally cure cancer is limited due not only to cancer heterogeneity but also to a poor understanding of the mechanisms underlying cancer recurrence. The heterogeneity of cells within the tumor plays a pivotal role as cancer cells possess different genetic makeup, proliferation rate and response to therapies. Among these cells, the population of quiescent cancer stem cells (qCSCs), also known as dormant cancer cells, has gained attention for their ability to reversibly arrest in a non-proliferating G0/earlyG1 phase to survive to cytotoxic insults. In this regard, these cells are considered to be responsible for cancer drug treatment failure in solid tumours such as CRC. Indeed, these qCSCs are more resistant to anti- proliferative drugs and can re-enter the cell cycle when the conditions are permissive, suggesting that they play an important role in tumour relapse. Although quiescent state is highly linked to drug resistance in cancer cells, due to their rarity and plasticity qCSCS remain mostly elusive and represent a challenging therapeutic target. The technologies available for identifying and studying this population are limited, time consuming and costly. The aim of this study was to develop a system that allow a fast and quantitative analysis of these cells by infecting them with the double vector Luc-RFP/p27- mVenus. With this vector, tumor cells were efficiently tracked by fluorescence and luciferase activity analyses, giving the possibility to extend its use in vivo bio-imaging and the analysis of 3D structures. To validate the system in vivo, immunocompromised mice were inoculated with CRCs-derived spheroids transduced with the double vector and then treated with chemotherapy drugs. Of note, by ex vivo flow cytometry analysis we demonstrated that chemotherapy determined an enrichment of qCSCs, consistently with the fact that these cells can resist to anti-proliferative agents. Moreover, by real-time analysis we demonstrated that these qCSCs acquired features of stemness, suggesting that quiescence emerges as a whole set of molecular properties covering multiple cellular processes. In conclusion, this innovative system allowed us not only to study the effects of new chemotherapeutics, but also to track and characterize the cell population responsible for cancer treatment failure, chemo-resistance and metastasis formation.