Dottorato in SCIENZE DELLA VITA

Titolo della tesi: A colorectal cancer stem cells (CSCs) biobank as a multi-level approach to drug sensitivity studies.

Abstract Cancer stem cells (CSCs), recently identified in the majority of solid tumors, are a subpopulation of slow-replicating cells that drive tumor growth, by generating a cascade of differentiated cells that actually constitutes the bulk of the tumor. CSCs are selectively resistant to antiproliferative and cytotoxic drugs; therefore, it is widely accepted that they represent one main determinant of tumor relapses following chemotherapy. Colorectal CSCs can be isolated from patient’s tumors, and grown in vitro in selective mediums in an undifferentiated state as spheroids. Upon injection into immunocompromised mice, they generate tumor xenografts that closely replicate the original parental tumor, in terms of both antigen expression and histological tissue organization. In the host institution, we have recently established a biobank of frozen colorectal CSCs derived from individual patient’s tumors [1]. Newly established cultures are routinely validated by i) patient-matching verification through STR (Short Tandem Repeats) analysis, ii) assessment of CSC capability to originate phenocopies of the original patient’s tumor upon xenografting into NSG mice, iii) analysis of expression of stem cell markers. CSCs are then molecularly characterized by whole exome sequencing (WES) and banked for future uses. This platform is a powerful tool for the screening of targeted therapies and for drug resistance molecular studies. During the last few years, it has allowed our group to assess and characterize the activity on colorectal CSCs of different antineoplastic agents, including CHK1-targeted therapy [2], novel NHERF1 inhibitors [3], and new chemical derivatives of fenretinide [4, 5]. In addition, it has allowed us to characterize the EMT-related transcription factor ZEB2 in CSCs quiescence and drug resistance [6]. Here in particular I report the results of a study on differential sensitivity to the anti-EGFR MoAb cetuximab, on different classes of Microsatellite Instable (MSI) versus Microsatellite Stable (MSS) colorectal cancer stem cells. MSI tumors represent a distinct group of colorectal cancers, characterized by deficiency in the mismatch repair machinery and by high mutation rate. By analyzing the effect of anti-EGFR MoAb cetuximab on a panel of cCSCs we observed that MSI strains display an increased resistance to anti-EGFR therapy as compared to MSS, irrespective of KRAS mutational status. Gene mutation analysis on MSI versus MSS CSCs revealed that MSI strains carry recurrent mutations within groups of genes coding for proteins with a role in RAS and calcium signaling, highlighting the role of a genomically unstable context in determining anti-EGFR resistance. Altogether, these results indicate a multifactorial origin of anti-EGFR resistance, which emerges as the effect of multiple events further than the KRAS mutation, targeting direct and indirect regulators of the EGFR pathway. Data have been published in [7]. References 1. De Angelis, M.L., et al., Cancer Stem Cell-Based Models of Colorectal Cancer Reveal Molecular Determinants of Therapy Resistance. Stem Cells Transl Med, 2016. 5(4): p. 511-23. 2. Manic, G., et al., CHK1-targeted therapy to deplete DNA replication-stressed, p53-deficient, hyperdiploid colorectal cancer stem cells. Gut, 2018. 67(5): p. 903-917. 3. Coluccia, A., et al., Drug Design and Synthesis of First in Class PDZ1 Targeting NHERF1 Inhibitors as Anticancer Agents. ACS Med Chem Lett, 2019. 10(4): p. 499-503. 4. Orienti, I., et al., A new bioavailable fenretinide formulation with antiproliferative, antimetabolic, and cytotoxic effects on solid tumors. Cell Death Dis, 2019. 10(7): p. 529. 5. Orienti, I., et al., A novel oral micellar fenretinide formulation with enhanced bioavailability and antitumour activity against multiple tumours from cancer stem cells. J Exp Clin Cancer Res, 2019. 38(1): p. 373. 6. Francescangeli, F., et al., A pre-existing population of ZEB2(+) quiescent cells with stemness and mesenchymal features dictate chemoresistance in colorectal cancer. J Exp Clin Cancer Res, 2020. 39(1): p. 2. 7. De Angelis, M.L., et al., Colorectal cancer spheroid biobanks: multi-level approaches to drug sensitivity studies. Cell Biol Toxicol, 2018. 34(6): p. 459-469.