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Digital archive of theses discussed at the University of Pisa

 

Thesis etd-10092023-102229


Thesis type
Tesi di specializzazione (5 anni)
Author
DI GANGI, ALESSANDRO
URN
etd-10092023-102229
Thesis title
SINGLE CELL BIOLOGY AS A NOVEL TOOL IN OSTEOSARCOMA RESEARCH: APPLICATION TO CHILDREN, ADOLESCENTS AND YOUNG ADULTS
Department
MEDICINA CLINICA E SPERIMENTALE
Course of study
PEDIATRIA
Supervisors
relatore Prof. Peroni, Diego
Keywords
  • adolescents
  • children
  • circulating tumor cells
  • DEPArray
  • osteosarcoma
  • young adults
Graduation session start date
03/11/2023
Availability
Withheld
Release date
03/11/2063
Summary
Osteosarcoma is a rare and malignant bone tumor, primarily affecting adolescents during their pubertal growth phase. It is characterized by a grim prognosis when accompanied by metastatic disease. Circulating tumor cells (CTCs) may play a significant role in the pathogenesis of osteosarcoma, but their specific function remains poorly understood.
Objectives: our primary objective was to implement a method for isolating CTCs in osteosarcoma patients using the DEPArray™ technology. We have then conducted downstream molecular analysis to elucidate their relationship with the primary tumor, metastasis, and clinical outcomes. Our secondary goal was to assess the efficacy of a droplet sequencing technique for single-cell RNA sequencing (scRNAseq) in identifying CTCs.
Methods: We enrolled pediatric patients from the prospective TOSCANO cohort, obtaining peripheral blood samples at the time of diagnosis (PRE) and prior to surgery (POST). Clinical data were collected, and patients were categorized as either good responders (GR) or poor responders (PR) based on histological necrosis (≥90% and < 90%, respectively).
We isolated peripheral blood mononuclear cells (PBMC) and performed physical-based enrichment using the microfluidic system Parsortix™. Subsequently, the cells were fixed and stained with DAPI (as a positive control) and antibodies against CD45 (as a negative control), cytokeratins 8/18/19, and EPCAM (indicative of an epithelial phenotype), as well as TWIST and vimentin (indicative of a mesenchymal phenotype). Single cells were then sorted using DEPAarray™. Whole genome amplification was performed on the individually sorted cells, followed by analysis of copy number alterations (CNAs).
In a landmark experiment, we gather data from a previous next-generation sequencing (NGS) custom panel applied on the diagnostic tissue specimen of OS4. We compared the revealed FLT4 point mutation among primary tumor, metastatic tissue and representative mesenchymal and epithelial CTCs with Sanger sequencing.
Furthermore, in a pilot experiment, we attempted to investigate CTCs within the PBMC collection using single-cell RNA sequencing (scRNAseq). We counted PBMCs (PRE) from OS4, obtained a small aliquot of 3000 cells, and enriched the remaining cells with Parsortix™. These two groups of cells were pooled and analyzed. Primary patient-derived osteosarcoma cells were analyzed in a separate run.
Results: enrollment is still open, preliminary data from 6 pediatric patients were considered. CTCs were efficiently isolated and both mesenchymal (M-CTCs) and epithelial circulating cells (E-CTCs) were found. A significant negative correlation was observed between E-CTCs(PRE) and maximum standard uptake value (SUVmax) at the diagnosis (r= -0.84; p=0.02) in PR patients. A border-line significant positive correlation was observed for POST-PRE variations (∆) in M-CTCs and progression free survival (PFS) duration (r= 0.80; p= 0.08). CNAs were only partially shared among primary tumors/metastasis and CTCs. In the landmark experiment with OS4 samples, we have confirmed that CTCs shared the known FLT4 point mutation (c.3971G>C) with the metastasis and the primary tumor. Heterozygous mutations was identified in all the sample with the exception of the representative M-CTC in which also a wild type karyotype was noted in contrast to OS4 primary tumor, metastasis and representative E-CTC. In relation to the secondary endpoint, scRNAseq revealed a possible cluster of tumor-educated platelets.
Conclusion: we successfully isolated and characterized CTCs in young osteosarcoma patients. The enumeration of CTCs proved valuable for clinical correlations, and the analysis of CNAs and point mutations indicated the shared characteristics of CTCs with the corresponding primary tumor or metastasis, although some differences of unknown significance were observed. In-droplet scRNAseq did not identify CTCs but provided additional information. Collectively, these observations underscore the efficiency of single-cell platforms in studying mesenchymal/epithelial plasticity in osteosarcoma, offering precious insights in clinical settings. While further studies are necessary to validate these preliminary findings, they hold the potential to shed new light on osteosarcoma pathogenesis and may have implications for therapeutic interventions.
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