The potential use of small extracellular vesicles as predictive marker in canine multicentric lymphoma

Author(s)

Taismara Kustro Garnica

• Document type: Master's Dissertation
• Press: Pirassununga.
• Institution: Universidade de São Paulo (USP). Faculdade de Zootecnica e Engenharia de Alimentos (FZE/BT)
• Defense date: 2020-05-28

Examining board members

Heidge Fukumasu; Andrigo Barboza de Nardi; Tiago Góss dos Santos

• Advisor: Heidge Fukumasu; Juliano Coelho da Silveira

Abstract

Lymphoma is the most common type of canine hematological malignancy, where the multicentric form is responsible for 75% of all cases. The standard treatment is the 19-week CHOP protocol, in which 85% of dogs achieve complete / partial response; however, a large portion of the disease relapses in a period of up to one year after diagnosis. The evaluation of the therapeutic response and the understanding of the mechanisms involved in the chemoresistance process have been the biggest challenges of canine lymphoma. Therefore, this work was divided into two main studies: In the first study, we investigated in vivo the potential of serum exosomes and their miRNAs as predictive markers for canine lymphoma. In the second study, we investigated in vitro the role of exosomes derived from chemoresistant cells in human and canine lineages of hematopoietic neoplasms. Twenty-two dogs were used for the first study (8 in Complete Remission and 14 in Disease Progression). The exosomes isolated from the dogs were evaluated for size and concentration and a 95 oncomir screening was performed on selected samples from patients with CR and PD. PD patients had a higher concentration of serum exosomes at the time of diagnosis than patients with CR (D0, P = 0.0277). The analysis of the ROC curve was significant for the concentration of exosomes to predict the response to CHOP (AUC = 0.8076, P = 0.0203) and overall survival (AUC = 0.8333, P = 0.0136). MiR-205 (P = 0.0384) and miR-222 (P = 0.0578) had a higher frequency in the CR group and mir-20a was more expressed in patients with CR (P = 0.085), while miR-93 was more expressed in patients with PD (P = 0.09). In the second study, we induced chemoresistance using the CHOP protocol in cells 3132 (canine B-cell lymphoma) and Jurkat cells (human T-cell leukemia). We have shown that resistant cells 3132-CR and Jurkat-CR have a slower doubling time compared to their respective naive cells. Cells 3132 and 3132-CR secreted a greater number of exosomes after chemotherapy (P = 0.0187), but there was no difference in the number of exosomes between strains (P = 0.7661). There was an increase in cell proliferation after treatment of the exosomes compared to the control for 3132 (P <0.001) and Jurkat (P <0.0035). However, there was no difference in proliferation comparing treatment using exosomes derived from native cells and those derived from CR cells for 3132 (P = 0.11) and Jurkat (P = 0.91). Cell proliferation was greater after 78 hours of treatment with exosomes for 3132 (P <0.001) and Jurkat (P <0.001). In conclusion, the results generated by these studies can trigger advances in veterinary oncology through the introduction of the liquid biopsy approach and the understanding of mechanisms of development of chemoresistance mediated by exosomes. (AU)

• FAPESP's process: 17/15406-3 - Extracellular vesicles as potencial predictive marker in canine multicentric lymphoma
• Grantee: Taismara Kustro Garnica
• Support Opportunities: Scholarships in Brazil - Master