P694: INVESTIGATING THE ROLE OF CELL ADHESION MOLECULES IN TUNNELING NANOTUBES FORMATION IN CHRONIC MYELOID LEUKEMIA MICROENVIRONMENT

Abstract

Background: The cross-talk and interactions between leukemia cells and their microenvironment play an important role in the development of drug resistance. Tunneling nanotubes (TNTs) represent a novel type of direct cell-cell communication way allowing for direct transfer of different types of cargo between distant cells. Our previous studies (Kolba et al., 2019) have shown that these structures are formed between bone marrow stromal HS-5 cells and chronic myeloid leukemia (CML) K562 cells. Significantly, TNT-mediated transfer of membrane vesicles from stromal to leukemic cells resulted in the stroma-mediated protection of CML cells from imatinib-induced apoptosis. Despite the efforts made to understand the molecular mechanisms regulating TNTs formation, they still remain unclear - especially in the leukemia microenvironment. We have shown that TNTs are formed between stromal and leukemic cells, following direct cell-cell interaction and cell dislodgement. Thus, we have focused our attention on cell adhesion molecules, as potential key players in tunneling nanotubes formation and TNT-mediated vesicles transfer between leukemic and stromal cells. Aims: The aim of presented studies was to elucidate the role of CD44 and ITGβ1 - one of the most important cell adhesion molecules, in TNTs formation between leukemic and stromal cells and TNT-mediated exchange of vesicles. Methods: To examine this, we established the HS-5 stromal cell line with silenced CD44 protein expression. Then, we co-cultured control (wt) or CD44-silenced HS-5 cells with leukemic K562 cells. For ITGβ1 studies, we used blocking anti-ITGβ1 antibody. Using confocal microscopy, three types of TNTs in co-cultures were counted: homotypic TNTs formed between stromal cells, homotypic TNTs formed between CML cells and TNTs formed between stromal and CML cells (heterotypic). Moreover, the TNT-mediated transfer of cellular vesicles from stromal to leukemic cells was assessed in these co-cultures using flow cytometry. Since CD44 expression increases in CML cells after co-culture with stroma, we examined its potential role in the drug resistance in CML acceptor cells. To do this, rhodamine-123 efflux assay was used. Results: We found that both, tunneling nanotubes number and uptake of cellular vesicles by leukemia, decreased after co-culture with CD44-silenced stromal cells and after blocking with anti-ITGβ1 antibody. Flow cytometry analyses of rhodamine-123 efflux showed significantly increased efflux pump activity in leukemic acceptor cells. Summary/Conclusion: Our data suggest that CD44 and ITGβ1 can play an important role in formation of tunneling nanotubes between leukemic and stromal cells and TNT-mediated cellular vesicles exchange. Moreover, CD44 presented in acceptor cells is involved in the development of drug resistance in leukemic cells, by promoting activity of efflux transporters.