Specific Circulating Microvesicles (cMVs) Populations in Paediatric ALL
Authors: Paderi Francesca , Frasson Chiara , Gelain Sabrina , Basso Giuseppe , Kronnie Geertruy 
Published: October 2019

Abstract
Abstract 933
Acute lymphoblastic leukaemia (ALL) genesis, development and maintenance, has been shown to involve a number of aspects. On the one hand along the line of the somatic mutation theory of cancer there are accumulating genetic aberration, on the other hand there are cytokines and growth factors that contribute to initiate and maintain the disease. The bone marrow microenvironment plays an important role in regulating the hematopoietic stem cell niche modulating normal haematopoiesis but also affecting leukaemogenic transformation. In the bone marrow microenvironment leukaemia cells receive signals that protect them and promote their survival or proliferation.
Horizontal transfer of cellular signals mediated by circulating microvesicles (cMVs) is an emerging mechanism of cell to cell interaction, also in the haematopoietic stem cell niche. These small plasma bodies can be isolated from peripheral blood and are characterised by varying sizes (exosomes of 50 –100 nm diameter and microparticles of 100–1000 nm), and MVs have been shown to transfer cellular messages from donor to target cells where they can modulate protein synthesis.
In this study we investigated MVs that were recovered from peripheral blood (PB) and the type of signal that could be transferred by MVs in paediatric ALL. Initially we characterised MVs isolated from the supernatant of three cell lines (ALL MHH CALL4, LAM ME-1, osteosarcoma MG-63). We then isolated MVs from plasma of 28 ALL patients at diagnosis before the onset of treatment and of 5 control samples. Plasma free of platelets obtained from peripheral blood (in EDTA) was subjected to a centrifugation step (16000 g, at 4°C for 1 hour). After recovery of the pellet MVs were stained by cytoplasmic dye carboxyfluorescein diacetate, succinimidyl ester (CFSE) and by monoclonal antibodies specific for CD markers of patients leukaemia blasts and cell lines. MVs were analysed by flow cytometry to investigate at cMVs surface antigen marker expression and physical parameters. We focused our analysis on a particle population of 150–400 nm in diameter.
MVs from cell lines showed surface antigen specific for the respective cell lineage and were shown to contain mRNA transcripts. Unexpectedly in most cases MVs isolated from patients did not show leukaemia cell surface antigens but were found to contain mRNA. MVs' RNA was extracted from 27 patients and from 4 controls by Trizol, according to manufacturer instructions. Standard RT-PCR was performed to detect osteoclast and osteoblast markers as RANK, RANKL and TNFα.
The presence at different extent of RANK/RANKL/TNFα within cMVs from patients and in none of control samples suggests that this pathway, mainly involved in bone remodelling, is contributing to signalling between the hematopoietic niche and the bone marrow microenvironment in leukaemia samples. Other reports have demonstrated that osteoclasts can induce the mobilization of haematopoietic progenitor cells under stress conditions. The role of the RANK/RANKL/TNFα messengers carried by PB derived MVs in ALL patients and how these might affect leukaemia progression and maintenance needs to be further explored. Moreover we found that some of the MVs isolated from patients known to carry specific translocations at blast cell levels e.g t(12:21) and t(1:19), presented the same genetic aberration at MVs mRNA transcripts. MVs from one of the above patients (t(12:21)positive at blasts) were also positive for blast cells surface antigen (CD19), as seen by flow cytometry. For the other patient (t(1:19)) we did not perform parallel flow cytometry analysis due to lack of material. These preliminary observations suggest that peripheral blood plasma contains MV populations with different features and functions contributing to transfer of messages to distal cells.
In conclusion this is the first study on cMV populations in paediatric ALL patients and our date seem to underscore a role for the bone microenvironment including cMVs in this disease.

Disclosures:
No relevant conflicts of interest to declare.