Belardinilli, Tamascia
Innovative 3D model for the establishment of primary paediatric brain tumour cultures: new platform for the preclinical study of immunotherapeutic approaches [Tesi di dottorato]

Gliomas, encompassing Low Grade (LGGs) and High Grade (HGGs) diseases are, overall, the most common solid tumors of the pediatric population. Children affected by LGGs that cannot be cured often struggle to obtain a complete remission of the disease, suffering from significant long-term sequelae. HGGs are instead associated with very grim outcomes and no innovative treatment so far has been able to change this poor prognosis. New treatment approaches, such as immunotherapy, are therefore needed for these patients. Gliomas, as all solid tumors, are characterized by a high structural complexity and a strongly immunosuppressive microenvironment, both responsible for the frequent failure of immunotherapeutic approaches. Such complexity is difficult to reproduce with common bidimensional (2D) systems, making the conventional tumor models unable to predict the real efficacy of these treatments. In order to overcome such hurdles and obtain more reliable tumor models, we developed an innovative fibrin-based hydrogel 3D model and tested it as platform to establish primary cultures of pediatric LGGs and HGGs. In particular, LGGs are otherwise extremely difficult to maintain in culture owing to the well-known activation of senescence pathways. To date, 37 samples of LGG were cultured in both 2D and 3D platforms, showing that the 3D-culture enables the stabilization of LGG. Cell lines identity was confirmed by short tandem repeats (STRs) and the immunohistochemical characterization (structure, H&E, Ki67, tumor and differentiation markers) in 3D cultures revealed phenotype and cellular organization closer to those observed in the onset sample, as compared to 2D. The 3D-cultured tumor cells showed a significantly (p =0,008) lower senescence rate than 2D. Furthermore, also 6 samples of HGG have been cultured and established as well. We therefore evaluated the antitumor activity of an innovative immunotherapeutic approach based on the use of γδ-T cells. These cells display properties of both the innate and the adaptive compartment, have a powerful cytotoxic activity, are able to recognize antigens independently from the HLA and display a negligible alloreactivity, resulting extremely attractive for clinical translation in particular for the establishment of an allogeneic “third-party T-cell bank” for ready for use off-the-shelf adoptive T-cell product. The clinical exploitation is limited, however, by the low percentage of circulating γδ-T cells and the lack, to date, of effective culture systems to expand them. We have developed a protocol to expand a clinically relevant number of polyclonal memory γδ-T cells. Artificial antigen presenting cells (aAPC) expressing CD86/4-1BBL/CD40L and cytomegalovirus-pp65 antigen were used to induce γδ-T cell expansion and activation. For increase the safety of our approach aAPCs has been further stable gene-modified with a suicide gene, inducible caspase 9, and then single cell cloned. The γδ-T cells expanded and expressed markers of activation and memory, maintaining a polyclonal phenotype (with a predominantly Vδ1 well known memory infiltrating population), negligible alloreactivity and potent antitumor capacity versus a broad range of malignancies. Lastly, we characterized the antitumor activity of expanded γδ-T cells, as well as of more conventional approaches (such as radiotherapy and chemotherapy), in 2D and in 3D-cultured tumors, showing that the first are significantly more sensitive to treatment. Expanded γδ-T cells showed a relevant antitumor activity in both 2D and 3D, the latter requiring longer cultures to obtain an antitumor control. These data suggest a more realistic estimation of the efficacy of such treatment by the 3D-platform, confirming it as a better model for biological and therapeutic studies. 

diritti: info:eu-repo/semantics/openAccess
In relazione con info:eu-repo/semantics/altIdentifier/hdl/11573/1349215
valutatori esterni: P. Nisticò, L. Ricci Vitiani
tutor esterno: F. Del Bufalo

Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD20018991