Primary Acute Lymphoblastic Leukemia Cells Create a Drug-Resistant Niche By Tunneling Nanotube Signaling with Mesenchymal Stromal Cells
Authors: Polak Roel , De Rooij Bob , Pieters Rob , den Boer Monique L. 
Published: October 2019

Abstract
Background: Acute lymphoblastic leukemia (ALL) cells reside in the bone marrow microenvironment, which nurtures and protects cells from chemotherapeutic drugs. Bidirectional signaling between leukemic cells and their niche is shown to be essential for these processes. A major gap in our knowledge is the lack of insight into the functional mechanism regulating and controlling this crosstalk. Tunneling nanotubes (TNTs) have recently been reported as a novel mode of communication between eukaryotic cells, which can facilitate the transport of several types of cellular cargo.
Objectives: This study aims to investigate the presence and functional importance of TNTs in the leukemic niche.
Results: Here, we show for the first time that TNT signaling occurs between primary patient-derived leukemic cells and primary mesenchymal stromal cells using flow cytometry and time-lapse confocal microscopy. TNTs form within minutes after the start of co-culture and efficiently transfer lipophilic carbocyanine dye DiI from ALL cells towards MSCs. Dye transfer was significantly reduced when TNT signaling was inhibited using three independent experimental setups: actin inhibition, mechanical disruption through gentle shaking of cell cultures, and physical separation using a 3.0 µm pore-sized transwell system (4-fold, 5-fold and 35-fold reduction, respectively). In reciprocal experiments we also observed dye transfer via TNTs from MSCs to BCP-ALL cells, but the magnitude was strikingly less (175-fold, p ≤ 0.001). In addition, we show that leukemic cells use TNTs to modulate their microenvironment by directing non-malignant stromal cells to produce pro-survival cytokines. We observed patient-specific cytokine signatures after co-culture of primary ALL cells with different primary MSCs. For example, IP10/CXCL10 levels increased more than 1000-fold when patient ALL#1 cells were co-cultured with primary MSCs, but were undetectable in co-cultures from patient ALL#2 cells. Induction of all cytokines was dependent on TNT signaling, as TNT inhibition significantly lowered cytokine production (p ≤ 0.001). Importantly, we show that TNT signaling is functionally important for leukemic cell survival and stroma-driven drug resistance. Leukemic cell viability was assessed by flow cytometry after staining with Annexin V, Propidium Iodide and CD19. Primary leukemic cell survival significantly increased in 5-day co-cultures with primary MSCs compared to mono-culture (p ≤ 0.001). When TNT formation was prevented by shaking of these co-cultures or by transwell conditions, the cell viability reduced 3.5- and 3.6-fold, respectively (p ≤ 0.001). TNT inhibition similarly reduced the survival of primary ALL cells in co-culture with MSCs during prednisolone exposure (p ≤ 0.01). Co-culture with primary MSCs also induced resistance to prednisolone 2.5-fold compared to mono-culture in a proliferative setting, using the BCP-ALL cell line NALM6. Inhibition of TNT formation by shaking or transwell conditions significantly reduced this effect: cells in co-culture were only 1.4- and 1.1-fold more resistant compared to mono-culture, respectively (p ≤ 0.01).
Conclusion: The presented study identifies TNT formation as a major regulator of interaction between ALL cells and their bone marrow niche, which facilitates signaling mainly from leukemic cells towards MSCs. This signaling drives the release of cytokines within the microenvironment. Disruption of tunneling nanotubes inhibits this release, diminishes the survival benefit that MSCs provide to primary ALL cells, and sensitizes ALL cells to the important anti-leukemic drug prednisolone. This observation gives insight into the pathogenesis of BCP-ALL and opens new avenues to develop more effective therapies that interfere with the leukemic niche.

Disclosures
No relevant conflicts of interest to declare.