Mitochondrial transfer in hematological malignancies

Extracellular vesicles (EVs) are a heterogeneous group of particles, between 15 nanometers and 10 microns in diameter, released by almost all cell types in physiological and pathological conditions, including tumors. EVs have recently emerged as particularly interesting informative vehicles, so that they could be considered a true “cell biopsy”. Indeed, EV cargo, including proteins, lipids, and nucleic acids, generally reflects the nature and status of the origin cells. In some cases, EVs are enriched of peculiar molecular cargo, thus suggesting at least a degree of specific cellular packaging. EVs are identified as important and critical players in intercellular communications in short and long distance interplays. Here, we examine the physiological role of EVs and their activity in cross-talk between bone marrow microenvironment and neoplastic cells in hematological malignancies (HMs). In these diseases, HM EVs can modify tumor and bone marrow microenvironment, making the latter “stronger” in supporting malignancy, inducing drug resistance, and suppressing the immune system. Moreover, EVs are abundant in biologic fluids and protect their molecular cargo against degradation. For these and other “natural” characteristics, EVs could be potential biomarkers in a context of HM liquid biopsy and therapeutic tools. These aspects will be also analyzed in this review.

Simple SummaryMitochondrial transfer plays a crucial role in the acquisition of drug resistance in multiple myeloma, but its exact mechanism is not yet clear; moreover, overcoming the drug resistance that it causes is also a major challenge. Our research on primary myeloma cell cultures reveals that mitochondrial transfer is bi-directional between bone marrow stromal cells and myeloma cells, occurring via tunneling nanotubes and partial cell fusion with extreme increases under the influence of chemotherapeutic drugs, whereupon survival and adenosine triphosphate levels increase, while mitochondrial superoxide levels decrease in myeloma cells. These changes and the elevation of superoxide levels in stromal cells are proportional to the amount of incorporated mitochondria derived from the other cell type and to the concentration of the used drug. Although the inhibition of mitochondrial transfer is limited between stromal and myeloma cells, the supportive effect of stromal cells can be effectively averted by influencing the tumor metabolism with an inhibitor of oxidative phosphorylation in addition to chemotherapeutics.Recently, it has become evident that mitochondrial transfer (MT) plays a crucial role in the acquisition of cancer drug resistance in many hematologic malignancies; however, for multiple myeloma, there is a need to generate novel data to better understand this mechanism. Here, we show that primary myeloma cells (MMs) respond to an increasing concentration of chemotherapeutic drugs with an increase in the acquisition of mitochondria from autologous bone marrow stromal cells (BM-MSCs), whereupon survival and adenosine triphosphate levels of MMs increase, while the mitochondrial superoxide levels decrease in MMs. These changes are proportional to the amount of incorporated BM-MSC-derived mitochondria and to the concentration of the used drug, but seem independent from the type and mechanism of action of chemotherapeutics. In parallel, BM-MSCs also incorporate an increasing amount of MM cell-derived mitochondria accompanied by an elevation of superoxide levels. Using the therapeutic antibodies Daratumumab, Isatuximab, or Elotuzumab, no similar effect was observed regarding the MT. Our research shows that MT occurs via tunneling nanotubes and partial cell fusion with extreme increases under the influence of chemotherapeutic drugs, but its inhibition is limited. However, the supportive effect of stromal cells can be effectively avoided by influencing the metabolism of myeloma cells with the concomitant use of chemotherapeutic agents and an inhibitor of oxidative phosphorylation.

A Novel Evidence That PNH Affected Cells Residing in Bone Marrow (BM) Due to Impaired Incorporation of CXCR4 and VLA-4 Into Membrane Lipid Rafts Show Defective SDF-1- and VCAM-1-Mediated Retention in BM What Leads to Their Increased Motility and Impaired Interaction with the BM Stem Cell Niches

PGC1α Driven Mitochondrial Biogenesis within the Bone Marrow Stromal Cells of the Acute Myeloid Leukemia Micro-Environment Is a Pre-Requisite for Mitochondrial Transfer to Leukemic Blasts

Mitochondria are not merely energy-producing organelles but also regulate metabolism, apoptosis, and inflammation. Recent studies have reported that mitochondria can be transferred between cells, and accumulating evidence suggests that this phenomenon is functionally relevant in the tumor context. Mitochondrial transfer is mediated by multiple routes such as tunneling nanotubes and extracellular vesicles. These pathways are regulated by Miro1/2, connexin 43, ICAM-1, VCAM-1, and intracellular reactive oxygen species. Within the tumor microenvironment, mitochondrial transfer from surrounding cells to tumor cells may serve as a mechanism by which tumor cells adapt to hostile metabolic conditions and evade therapeutic pressure. Furthermore, mitochondrial transfer from tumor cells to T cells in the tumor microenvironment reportedly impairs antitumor immunity. Based on these findings, novel therapeutic strategies targeting mitochondrial transfer are under investigation. Future challenges include the development of specific and safe methods to manipulate mitochondrial transfer invivo. Understanding mitochondrial transfer and its regulation may offer new avenues to overcome resistance and improve cancer outcomes.

Background:Neoplastic cells and bone marrow (BM) stromal cells interact intimately in the marrow and communicate with each other in numerous ways such as paracrine signaling via cytokines and chemokines, cell‐to‐cell interactions through tunneling nanotubes (TNTs) or gap junctions, and the secretion and incorporation of microvesicles (MVs). As the acquisition of cancer drug resistance was associated with mitochondrial transfer in several malignancies, we investigated the possibility of a similar phenomenon in multiple myeloma (MM) using an in vitro stromal cell–malignant plasma cell co‐culture system.Aims:Our primary goal was to establish whether mitochondrial transfer occurs between BM stromal cells and primary myeloma cells, and if so, exactly by what mechanism. We also wanted to clarify if this phenomenon could contribute to the drug resistance of myeloma cells.Methods:Mitochondrial exchange between BM stromal cells and myeloma cells was quantified by flow cytometry and visualized by confocal laser scanning microscopy. Cellular toxicity of different drugs was assessed by high throughput screening.Results:We found a relatively fast, bidirectional mitochondrial transfer between BM stromal cells and malignant plasma cells. As mitochondria can be transferred through TNTs, gap junctions, or via MVs, we selectively inhibited these pathways to dissect the exact mechanisms involved. According to our results, only cytochalasin D, a potential blocker of TNT formation, was able to reduce significantly the amount of transferred mitochondria in a dose‐dependent manner; however, the transmission of mitochondria has also been slightly reduced by inhibiting the MV uptake or gap junctions, suggesting a dominant but non‐exclusive role of TNTs in this process. In further experiments we demonstrated that stromal cells accumulate substantial amounts of mitochondria from myeloma cells delivered by MVs, whereas myeloma cells incorporate much less MVs and thus less mitochondria from stromal cells. Confocal microscopic analysis also confirmed bidirectional mitochondrial transport between stromal and myeloma cells through TNTs. Finally, we evaluated the possible contribution of stromal cell‐derived and myeloma cell‐derived mitochondria to the drug resistance of malignant plasma cells. Several drugs including apoptosis inducers, proteasome inhibitors, and epigenetic modifiers were tested, and it was clearly demonstrated that drug treatment always enhances the transfer of mitochondria from stromal cells to myeloma cells in a dose‐dependent manner (see figure for data on carfilzomib).Summary/Conclusion:Our cytotoxic assays demonstrated that myeloma cells are more resistant to drug treatment in co‐cultures than alone, and it is most likely caused by bi‐directional mitochondrial transfer. Our results provide additional clues for understanding the biological processes that underlie cell adhesion‐mediated drug resistance upon myeloma therapy.imageThis work was supported by grant NVKP_16–1–2016–0005 from the NKFIA, Hungary

Targeting mitochondrial transfer as a promising therapeutic strategy.

Mitochondrial transfer (MT) is a biological process that allows a donor cell to horizontally share its own mitochondria with a recipient cell. Mitochondria are highly dynamic membrane-bound sub-cellular organelles prominently involved in the regulation of the cell energy balance, calcium homeostasis, and apoptotic machinery activation. They physiologically undergo fusion and fission processes in response to the cell requirement, with a continuous morphological re-arrangement. This structural and functional plasticity is at the basis of the MT, described in tissue regeneration, cardiac and neurological diseases, as well as in cancer. Here, the MT has been observed in the tumor micro-environment (TME) from the adipose-derived stem cells (ASCs) to the cancer cells, eventually reverting the lack of the mitochondria respiration function, or enhancing their motility and drug resistance. In this chapter, we outline some key protocols for evaluating this exciting phenomenon of MT. These methodological and technical approaches are very important, considering all the limitations that scientists constantly face, especially in this field of the research.

Intercellular communication is essential for multicellular tissue vitality and homeostasis. We show that healthy cells message protective signals through direct cell–cell connections to adjacent DNA–damaged cells in a microtubule–dependent manner. In DNA–damaged cells, mitochondria restoration is facilitated by fusion with undamaged mitochondria from healthy cells and their DNA damage repair is optimized in presence of healthy cells. Both, mitochondria transfer and intercellular signaling for an enhanced DNA damage response are critically regulated by the activity of the DNA repair protein ataxia telangiectasia mutated (ATM). These healthy–to–damaged prosurvival processes sustain normal tissue integrity and may be exploitable for overcoming resistance to therapy in diseases such as cancer.

Burkitt lymphoma (BL) is a highly aggressive form of non-Hodgkin's B-cell lymphoma. Currently, multi-agent chemotherapy regimens are being used to significantly improve cure rates and achieve complete remissions in BL patients. However, drug resistance can often occur within 6 months in BL patients, contributing to poor prognosis. Mounting evidence suggests that cell adhesion-mediated drug resistance (CAM-DR), caused by the interaction between the bone marrow microenvironment and tumour cells may play an important role in drug resistance to chemotherapy. However, the molecular mechanism underlying CAM-DR in BL has not been identified yet. In this study, we investigated the molecular mechanism responsible for CAM-DR in BL cells. We also examined the therapeutic targets of CAM-DR in BL cells and found CD49d and CD49e to be the important adhesion molecules involved. However, CD49a, CD49b, CD11a, CD29, CD18, and CD61 were not found to be associated with CAM-DR in BL cells. Furthermore, we clarified that CD49d- and CD49e-mediated CAM-DR could be attributed to an increase in the expression of B cell leukemia-xL (Bcl-xL) and survivin proteins, and a decrease in the expression of Bcl-2 associated X (Bax), Bcl-2 interacting mediator (Bim) and p53 upregulated modulator of apoptosis (PUMA) proteins via nuclear factor kappaB (NF-κB) activation. In addition, bortezomib was found to overcome CAM-DR in BL cells by inhibiting NF-κB. Thus, bortezomib may have potential clinical applications in the treatment of CD49d- and CD49e-mediated CAM-DR in BL patients.

As hematopoietic stem cells can differentiate into all hematopoietic lineages, mitigating the damage to hematopoietic stem cells is important for recovery from overdose radiation injury. Cells in bone marrow microenvironment are essential for hematopoietic stem cells maintenance and protection, and many of the paracrine mediators have been discovered in shaping hematopoietic function. Several recent reports support exosomes as effective regulators of hematopoietic stem cells, but the role of osteoblast derived exosomes in hematopoietic stem cells protection is less understood. Here, we investigated that osteoblast derived exosomes could alleviate radiation damage to hematopoietic stem cells. We show that intravenous injection of osteoblast derived exosomes promoted WBC, lymphocyte, monocyte and hematopoietic stem cells recovery after irradiation significantly. By sequencing osteoblast derived exosomes derived miRNAs and verified in vitro, we identified miR-21 is involved in hematopoietic stem cells protection via targeting PDCD4. Collectively, our data demonstrate that osteoblast derived exosomes derived miR-21 is a resultful regulator to radio-protection of hematopoietic stem cells and provide a new strategy for reducing radiation induced hematopoietic injury.

BackgroundBone marrow-derived mesenchymal stromal cells (BM-MSCs) are capable of repairing wounded lung epithelial cells by donating cytoplasmic material and mitochondria. Recently, we characterized two populations of human lung-derived mesenchymal stromal cells isolated from digested parenchymal lung tissue (LT-MSCs) from healthy individuals or from lung transplant recipients’ bronchoalveolar lavage fluid (BAL-MSCs). The aim of this study was to determine whether LT-MSCs and BAL-MSCs are also capable of donating cytoplasmic content and mitochondria to lung epithelial cells.MethodsCytoplasmic and mitochondrial transfer was assessed by co-culturing BEAS2B epithelial cells with Calcein AM or Mitotracker Green FM-labelled MSCs. Transfer was then measured by flow cytometry and validated by fluorescent microscopy. Molecular inhibitors were used to determine the contribution of microtubules/tunnelling nanotubes (TNTs, cytochalasin D), gap junctions (carbenoxolone), connexin-43 (gap26) and microvesicles (dynasore).ResultsF-actin microtubules/TNTs extending from BM-MSCs, LT-MSCs and BAL-MSCs to bronchial epithelial cells formed within 45 minutes of co-culturing cells. Each MSC population transferred a similar volume of cytoplasmic content to epithelial cells. Inhibiting microtubule/TNTs, gap junction formation and microvesicle endocytosis abrogated the transfer of cytoplasmic material from BM-MSCs, LT-MSCs and BAL-MSCs to epithelial cells. In contrast, blocking connexin-43 gap junction formation had no effect on cytoplasmic transfer. All MSC populations donated mitochondria to bronchial epithelial cells with similar efficiency. Mitochondrial transfer was reduced in all co-cultures after microtubule/TNT or endocytosis inhibition. Gap junction formation inhibition reduced mitochondrial transfer in BM-MSC and BAL-MSC co-cultures but had no effect on transfer in LT-MSC co-cultures. Connexin-43 inhibition did not impact mitochondrial transfer. Finally, bronchial epithelial cells were incapable of donating cytoplasmic content or mitochondria to any MSC population.ConclusionSimilar to their bone marrow counterparts, LT-MSCs and BAL-MSCs can donate cytoplasmic content and mitochondria to bronchial epithelial cells via multiple mechanisms. Given that BM-MSCs utilize these mechanisms to mediate the repair of damaged bronchial epithelial cells, both LT-MSCs and BAL-MSCs will probably function similarly.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0354-8) contains supplementary material, which is available to authorized users.

Mitochondria are essential organelles involved in the maintenance of cell growth and function, and have been investigated as therapeutic targets in various diseases. Recent studies have demonstrated that direct mitochondrial transfer can restore cellular functions of cells with inherited or acquired mitochondrial dysfunction. However, previous mitochondrial transfer methods are inefficient and time-consuming. Here, we developed a simple and easy mitochondrial transfer protocol using centrifugation, which can be applied to any cell type. By our simple centrifugation method, we found that the isolated mitochondria could be successfully transferred into target cells, including mitochondrial DNA-deleted Rho0 cells and dexamethasone-treated atrophic muscle cells. We found that mitochondrial transfer normalised ATP production, mitochondrial membrane potential, mitochondrial reactive oxygen species level, and the oxygen consumption rate of the target cells. Furthermore, delivery of intact mitochondria blocked the AMPK/FoxO3/Atrogene pathway underlying muscle atrophy in atrophic muscle cells. Taken together, this simple and rapid mitochondrial transfer method can be used to treat mitochondrial dysfunction-related diseases.

Within the central nervous system (CNS), mitochondria serve as vital energy sources for neurons, glial cells, and vascular functions, maintaining intracellular metabolic balance. Recent studies involving cellular models, rodents, and humans reveal that metabolically active mitochondria can be released into the extracellular space, playing roles in intercellular communication within the CNS. When taken up by neurons, these extracellular mitochondria may provide neuroprotective effects. Conversely, damaged mitochondria and their released components during severe tissue injury or inflammation can contribute to neurodegenerative processes. Thus, mitochondria secreted under pathological conditions in the CNS hold promise as biomarkers indicative of recovery. Additionally, transplantation of external mitochondria shows potential as a therapeutic approach for various CNS disorders. This mini review focuses on recent advances in the transfer of mitochondria between cells, the use of extracellular mitochondria as biomarkers, and the prospects of mitochondria transplantation from experimental research to clinical application, particularly in diseases like stroke.

HOXB7 Overexpression in Mesenchymal Cells Stimulates the Production of Pro-Angiogenic Molecules: Potential Role in Multiple Myeloma Associated Angiogenesis