Evolution and progression of adrenocortical carcinoma: the potential role of the adipose microenvironment and the isolation and characterization of circulating tumor cells

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy affecting the adrenal cortex, presenting high heterogeneity and aggressiveness, often with an unfavorable outcome. Despite several novel biomarkers of diagnostic and prognostic relevance have been identified, the molecular mechanism underlying the pathology has still to be fully elucidated and the available therapeutic options show limited specificity and efficacy. Molecular alterations, including driver gene mutations, epigenetic alterations and dysregulation of signaling pathways related to cell survival, growth and proliferation, play a crucial role in determine malignancy. However, this cannot exhaustively enucleate the mechanisms by which cancer cells acquire the ability to migrate and metastasize to distant sites, thus resulting in cancer progression. Tumor microenvironment constitutes the ideal soil to establish a dynamic crosstalk between tumor and stromal cells, inducing reciprocal metabolic and functional alterations. In particular, adipocytes and adipose progenitors have been demonstrated to play a pivotal role in supporting tumor growth and progression. Since the adrenal glands present a substantial component of adipose tissue, the adipose microenvironment may be involved in the mechanisms underlying adrenal tumorigenesis and cancer evolution. The first objective of this thesis was to was reproduce an in vitro tumor microenvironment by co-culturing the adrenocortical cancer cells NCI-H295R with cells of the adipose lineage, particularly adipose-derived stem cells (ASCs) and in vitro differentiated adipocytes, in order to evaluate the reciprocal paracrine effects on cell behavior. This was achieved by using a culturing system in which the two cell types were physically separated but a constant exchange of soluble factors was assured by the presence of a porous membrane. Through this method we demonstrated that an active crosstalk was established between the two cell types, leading to cancer cell increased proliferation and migration ability, but also to a altered morphological and functional features in adipose cells, acquiring a phenotype more prone to sustain cancer growth. Studying the dynamics of such interactions would help in elucidating adrenocortical cancer biology and in developing more specific and effective treatments. Identifying specific features of more aggressive cancer cells could also allow to monitor tumor progression, preventing tumor dissemination and metastasis. In this scenario, the "liquid biopsy" may represent the possibility to follow tumor evolution: the detection and characterization of circulating tumor cells (CTCs) in blood samples of cancer patients represents a promising way to profile cancer disease complexity at any stage of tumor progression, by a minimally-invasive procedure. As shown in the present study, CTCs can be isolated and identified for their cancer and adrenal nature in blood samples of ACC patients. Moreover, an experimental workflow was designed in order to compare CTC genetic profile to that of the primary tumor. In particular, DNA extraction and amplification methods were tested on CTC samples to assess feasibility for downstream applications, such as next generation sequencing and digital-droplet PCR. The identification of specific traits characterizing the pool of cancer cells retaining the metastatic potential would be particularly meaningful in advanced adrenocortical carcinoma, aiding to monitoring disease evolution and to develop therapeutic strategies patient-centered.