Tunneling nanotube (TNT) formation is downregulated by cytarabine and NF-κB inhibition in acute myeloid leukemia (AML)

Abstract

Acute myeloid leukemia (AML) is a bone marrow derived blood cancer where intercellular communication in the leukemic bone marrow participates in disease development, progression and chemoresistance. Tunneling nanotubes (TNTs) are intercellular communication structures involved in transport of cellular contents and pathogens, also demonstrated to play a role in both cell death modulation and chemoresistance. Here we investigated the presence of TNTs by live fluorescent microscopy and identified TNT formation between primary AML cells and in AML cell lines. We found that NF-κB activity was involved in TNT regulation and formation. Cytarabine downregulated TNTs and inhibited NF-κB alone and in combination with daunorubicin, providing additional support for involvement of the NF-κB pathway in TNT formation. Interestingly, daunorubicin was found to localize to lysosomes in TNTs connecting AML cells indicating a novel function of TNTs as drug transporting devices. We conclude that TNT communication could reflect important biological features of AML that may be explored in future therapy development.

Tyrosine kinase inhibitors and interferon‐α increase tunneling nanotube (TNT) formation and cell adhesion in chronic myeloid leukemia (CML) cell lines

“…Since the tyrosine kinase ABL1 and the oncogenic fusion protein BCR‐ABL1 include distinct F‐ and G‐actin binding domains with actin bundling activity, we examined various BCR‐ABL1 expressing cells with respect to TNT formation. BCR‐ABL1 positive CML cells, cell lines, and patient‐derived cells, displayed limited numbers or absence of TNT formation compared to acute myeloid leukemia cells, stromal cells and other cancer cells . TNT formation between cells in vitro is highly dependent on adherence, and culturing leukocytes on a supportive layer of mesenchymal stem cells (MSCs) or fibronectin increases TNT formation .…”

“…The following were used for actin and membrane staining: Alexa Fluor 350-conjugated phalloidin and wheat germ agglutinin (WGA)-Alexa Fluor 594 or 488 (Invitrogen) as previously described. 31 TKIs: imatinib, nilotinib, bosutinib, ponatinib, and dasatinib (Selleckchem). IFNα (Intron A from MSD), Cytochalasin D (Sigma-Aldrich), doxycycline (Doxyferm, Nordic Drugs AB, Limhamn), puromycin (Sigma-Aldrich), Bovine serum albumin (BSA) fraction V (Roche), fibronectin (Sigma-Aldrich).…”

“…TNTs were distinguished from cytoplasmic bridges, which appear following cell division, by the lack of a midbody clearly visible by differential interference contrast and/or staining of cellular membranes. 31 8-well µ-slides (Ibidi GmbH) were pre-coated with fibronectin (10 µg/mL, F2006, Sigma-Aldrich) for 30 minutes at 37°C before washed with saline. 70 000 cells were seeded per well and incubated overnight under physiological conditions.…”

“…They are involved in cell‐to‐cell interaction and intercellular transport of organelles and pathogens such as virus and bacteria . Leukocytes, their leukemic counterparts and bone marrow stromal cells have all been reported to form TNTs in vitro . TNT is proposed to be a mechanism for chemo resistance by transport of oncoproteins between T and B cells as well as in colon cancer cells, by transfer of mitochondria from endothelial cells to chemotherapy exposed cancer cells, or by induced drug‐efflux in aggressive forms of pancreatic carcinoma .…”

“…16,[18][19][20][21][22][23][24] Leukocytes, their leukemic counterparts and bone marrow stromal cells have all been reported to form TNTs in vitro. [25][26][27][28][29][30][31][32] TNT is proposed to be a mechanism for chemo resistance by transport of oncoproteins between T and B cells as well as in colon cancer cells, by transfer of mitochondria from endothelial cells to chemotherapy exposed cancer cells, or by induced drug-efflux in aggressive forms of pancreatic carcinoma. [33][34][35][36][37][38] The impact of TNTs in vivo is so far not well characterized, but they have been described to connect myeloid cells in the cornea of mouse 39,40 and in fresh resected tumor samples from patients with malignant pleural mesothelioma and lung adenocarcinoma.…”