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Macrophage polarization impacts tunneling nanotube formation and intercellular organelle trafficking.

ABSTRACT: Tunneling nanotubes (TNTs) are cellular extensions enabling cytosol-to-cytosol intercellular interaction between numerous cell types including macrophages. Previous studies of hematopoietic stem and progenitor cell (HSPC) transplantation for the lysosomal storage disorder cystinosis have shown that HSPC-derived macrophages form TNTs to deliver cystinosin-bearing lysosomes to cystinotic cells, leading to tissue preservation. Here, we explored if macrophage polarization to either proinflammatory M1-like M(LPS/IFN?) or anti-inflammatory M2-like M(IL-4/IL-10) affected TNT-like protrusion formation, intercellular transport and, ultimately, the efficacy of cystinosis prevention. We designed new automated image processing algorithms used to demonstrate that LPS/IFN? polarization decreased bone marrow-derived macrophages (BMDMs) formation of protrusions, some of which displayed characteristics of TNTs, including cytoskeletal structure, 3D morphology and size. In contrast, co-culture of macrophages with cystinotic fibroblasts yielded more frequent and larger protrusions, as well as increased lysosomal and mitochondrial intercellular trafficking to the diseased fibroblasts. Unexpectedly, we observed normal protrusion formation and therapeutic efficacy following disruption of anti-inflammatory IL-4/IL-10 polarization in vivo by transplantation of HSPCs isolated from the Rac2^-/- mouse model. Altogether, we developed unbiased image quantification systems that probe mechanistic aspects of TNT formation and function in vitro, while HSPC transplantation into cystinotic mice provides a complex in vivo disease model. While the differences between polarization cell culture and mouse models exemplify the oversimplicity of in vitro cytokine treatment, they simultaneously demonstrate the utility of our co-culture model which recapitulates the in vivo phenomenon of diseased cystinotic cells stimulating thicker TNT formation and intercellular trafficking from macrophages. Ultimately, we can use both approaches to expand the utility of TNT-like protrusions as a delivery system for regenerative medicine.

SUBMITTER: Goodman S

PROVIDER: S-EPMC6787037 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Macrophage polarization impacts tunneling nanotube formation and intercellular organelle trafficking.

Goodman Spencer S Naphade Swati S Khan Meisha M Sharma Jay J Cherqui Stephanie S

Scientific reports 20191010 1

Tunneling nanotubes (TNTs) are cellular extensions enabling cytosol-to-cytosol intercellular interaction between numerous cell types including macrophages. Previous studies of hematopoietic stem and progenitor cell (HSPC) transplantation for the lysosomal storage disorder cystinosis have shown that HSPC-derived macrophages form TNTs to deliver cystinosin-bearing lysosomes to cystinotic cells, leading to tissue preservation. Here, we explored if macrophage polarization to either proinflammatory M ...[more]

PMID: 31601865

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Project description:Disruption of intercellular communication within tumors is emerging as a novel potential strategy for cancer-directed therapy. Tumor-Treating Fields (TTFields) therapy is a treatment modality that has itself emerged over the past decade in active clinical use for patients with glioblastoma and malignant mesothelioma, based on the principle of using low-intensity alternating electric fields to disrupt microtubules in cancer cells undergoing mitosis. There is a need to identify other cellular and molecular effects of this treatment approach that could explain reported increased overall survival when TTFields are added to standard systemic agents. Tunneling nanotube (TNTs) are cell-contact-dependent filamentous-actin-based cellular protrusions that can connect two or more cells at long-range. They are upregulated in cancer, facilitating cell growth, differentiation, and in the case of invasive cancer phenotypes, a more chemoresistant phenotype. To determine whether TNTs present a potential therapeutic target for TTFields, we applied TTFields to malignant pleural mesothelioma (MPM) cells forming TNTs in vitro. TTFields at 1.0 V/cm significantly suppressed TNT formation in biphasic subtype MPM, but not sarcomatoid MPM, independent of effects on cell number. TTFields did not significantly affect function of TNTs assessed by measuring intercellular transport of mitochondrial cargo via intact TNTs. We further leveraged a spatial transcriptomic approach to characterize TTFields-induced changes to molecular profiles in vivo using an animal model of MPM. We discovered TTFields induced upregulation of immuno-oncologic biomarkers with simultaneous downregulation of pathways associated with cell hyperproliferation, invasion, and other critical regulators of oncogenic growth. Several molecular classes and pathways coincide with markers that we and others have found to be differentially expressed in cancer cell TNTs, including MPM specifically. We visualized short TNTs in the dense stromatous tumor material selected as regions of interest for spatial genomic assessment. Superimposing these regions of interest from spatial genomics over the plane of TNT clusters imaged in intact tissue is a new method that we designate Spatial Profiling of Tunneling nanoTubes (SPOTT). In sum, these results position TNTs as potential therapeutic targets for TTFields-directed cancer treatment strategies. We also identified the ability of TTFields to remodel the tumor microenvironment landscape at the molecular level, thereby presenting a potential novel strategy for converting tumors at the cellular level from 'cold' to 'hot' for potential response to immunotherapeutic drugs.

Dataset Information

Macrophage polarization impacts tunneling nanotube formation and intercellular organelle trafficking.

Publications

Macrophage polarization impacts tunneling nanotube formation and intercellular organelle trafficking.

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