Oral Presentation Australia and New Zealand Society for Extracellular Vesicles Conference 2025

From Little Things, Big Things Grow: Investigating The Role Of Extracellular Vesicle Populations In CRLF2 Rearranged Acute Lymphoblastic Leukaemia (121567)

Maxim Buckley 1 , Elyse C Page 1 , Fiona Whelan 2 , Cherie Blenkiron 3 , Deborah L White 1 , Laura N Eadie 1
  1. Adelaide Medical School, The University of Adelaide/SAHMRI, Adelaide, SA, Australia
  2. Adelaide Microscopy, The University of Adelaide, Adelaide, SA, Australia
  3. Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand

Intro/aims: Extracellular vesicles (EVs) are lipid bodies produced by all cells. Medium EVs (mEVs - 100-1000 nm, formed by budding of cell membranes) and small EVs (sEVs – 50-150 nm, formed intracellularly), carry nucleic acids and proteins, and may be involved in transfer of oncogenic characteristics. We investigated if either of these EV populations could transfer the constitutively active CRLF2 p.F232C receptor (TSLPR) and subsequently induce leukaemic transformation.

Methods: Parental Ba/F3 cells were treated daily for 20 days with either mEVs or sEVs derived from CRLF2 p.F232C Ba/F3 cells in the presence of IL-3, to mimic the bone marrow microenvironment. Flow cytometry monitored TSLPR expression in Ba/F3 cells and detected TSLPR on mEVs. mEV-treated Ba/F3 cells were FACS sorted for TSLPR+ cells after 20 days of treatment. Cell Titre Glo determined growth in the absence of IL-3. Phosphoflow cytometry investigated changes in STAT5 and ERK phosphorylation.

Results: mEV-treated Ba/F3 cells demonstrated significantly increased TSLPR expression compared to vehicle (MFI=1.225 vs 1.0, p<0.0001) and sEV-treated (MFI=1.225 vs 1.035, p<0.0001) cells after 20 days of treatment. TSLPR was detected on mEVs analysed by flow cytometry. A purified population of TSLPR+ Ba/F3 cells demonstrated cytokine-independent growth that was significantly increased compared to parental control (day 4 fold change=2516 vs. 48.86, p=0.0104), with a significant increase in phosphorylated-STAT5 and ERK compared to parental control (p=0.008; p=0.0052 respectively). A significant temporal change in TSLPR expression was observed at week 1 vs. 3 (p=0.0457) but broadly remained consistent.

Conclusion: For the first time in an ALL setting, we have demonstrated horizontal transfer of protein and genomic material via mEVs. This horizontal transfer induces leukaemic transformation, altering growth and intracellular signalling profiles. These results indicate a broader role for EVs in leukaemia and have potential implications for leukaemia pathogenesis and relapse.