Yevgeniy Romin

Tunneling Nanotubes Provide a Unique Conduit for Intercellular Transfer of Cellular Contents in Human Malignant Pleural Mesothelioma

Tunneling nanotubes are long, non-adherent F-actin-based cytoplasmic extensions which connect proximal or distant cells and facilitate intercellular transfer. The identification of nanotubes has been limited to cell lines, and their role in cancer remains unclear. We detected tunneling nanotubes in mesothelioma cell lines and primary human mesothelioma cells. Using a low serum, hyperglycemic, acidic growth medium, we stimulated nanotube formation and bidirectional transfer of vesicles, proteins, and mitochondria between cells. Notably, nanotubes developed between malignant cells or between normal mesothelial cells, but not between malignant and normal cells. Immunofluorescent staining revealed their actin-based assembly and structure. Metformin and an mTor inhibitor, Everolimus, effectively suppressed nanotube formation. Confocal microscopy with 3-dimensional reconstructions of sectioned surgical specimens demonstrated for the first time the presence of nanotubes in human mesothelioma and lung adenocarcinoma tumor specimens. We provide the first evidence of tunneling nanotubes in human primary tumors and cancer cells and propose that these structures play an important role in cancer cell pathogenesis and invasion.

Tunneling Nanotubes: A new paradigm for studying intercellular communication and therapeutics in cancer.

Tunneling nanotubes are actin-based cytoplasmic extensions that function as intercellular channels in a wide variety of cell types.There is a renewed and keen interest in the examination of modes of intercellular communication in cells of all types, especially in the field of cancer biology. Tunneling nanotubes –which in the literature have also been referred to as “membrane nanotubes,” “’intercellular’ or ‘epithelial’ bridges,” or “cytoplasmic extensions” – are under active investigation for their role in facilitating direct intercellular communication. These structures have not, until recently, been scrutinized as a unique and previously unrecognized form of direct cell-to-cell transmission of cellular cargo in the context of human cancer. Our recent study of tunneling nanotubes in human malignant pleural mesothelioma and lung adenocarcinomas demonstrated efficient transfer of cellular contents, including proteins, Golgi vesicles, and mitochondria, between cells derived from several well-established cancer cell lines. Further, we provided effective demonstration that such nanotubes can form between primary malignant cells from human patients. For the first time, we also demonstrated the in vivo relevance of these structures in humans, having effectively imaged nanotubes in intact solid tumors from patients. Here we provide further analysis and discussion on our findings, and offer a prospective ‘road map’ for studying tunneling nanotubes in the context of human cancer. We hope that further understanding of the mechanisms, methods of transfer, and particularly the role of nanotubes in tumor-stromal cross-talk will lead to identification of new selective targets for cancer therapeutics.

Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells.

Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48 h; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells.

Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection

Immunofluorescent staining is an informative tool that is widely used in basic research. Automation of immunostaining improves reproducibility and quality of the results. Up to now, use of automation in immunofluorescent staining was mostly limited to one marker. Here we present tyramide signal amplification based method of multiple marker immunofluorescent detection, including detection of antibodies, raised in the same species, in tissue sections and cultured cells. This method can be beneficial for both basic and clinical research.

Targeted fibrillar nanocarbon RNAi treatment of acute kidney injury

Fibrillar carbon nanotubes simultaneously deliver two small interfering RNAs, which safely prevent acute kidney injury and prolong survival in mice. Double trouble for kidney toxicity The kidneys can be damaged by drugs, such as antibiotics and chemotherapy, as well as by surgery, which robs the organs of oxygen. To prevent injury, Alidori et al. devised a nanomedicine treatment approach that delivers two small interfering RNAs (siRNAs) to the main cells of the kidney, the renal proximal tubule cells. siRNAs targeting Mep1b and Trp53 were attached to fibrillar carbon nanotubes and delivered simultaneously to mice before drug-induced kidney insult. With such RNA interference, the kidney cells could not produce meprin-1β and p53—two key proteins involved in kidney injury; the mice lived longer and remained injury-free, but only if given both siRNAs. The nanotube/siRNA complexes were also safe and had favorable pharmacokinetics in monkeys. The next steps will be testing the dual siRNAs in other animal models of kidney injury. RNA interference has tremendous yet unrealized potential to treat a wide range of illnesses. Innovative solutions are needed to protect and selectively deliver small interfering RNA (siRNA) cargo to and within a target cell to fully exploit siRNA as a therapeutic tool in vivo. Herein, we describe ammonium-functionalized carbon nanotube (fCNT)–mediated transport of siRNA selectively and with high efficiency to renal proximal tubule cells in animal models of acute kidney injury (AKI). fCNT enhanced siRNA delivery to tubule cells compared to siRNA alone and effectively knocked down the expression of several target genes, including Trp53, Mep1b, Ctr1, and EGFP. A clinically relevant cisplatin-induced murine model of AKI was used to evaluate the therapeutic potential of fCNT-targeted siRNA to effectively halt the pathogenesis of renal injury. Prophylactic treatment with a combination of fCNT/siMep1b and fCNT/siTrp53 significantly improved progression-free survival compared to controls via a mechanism that required concurrent reduction of meprin-1β and p53 expression. The fCNT/siRNA was well tolerated, and no toxicological consequences were observed in murine models. Toward clinical application of this platform, fCNTs were evaluated for the first time in nonhuman primates. The rapid and kidney-specific pharmacokinetic profile of fCNT in primates was comparable to what was observed in mice and suggests that this approach is amenable for use in humans. The nanocarbon-mediated delivery of siRNA provides a therapeutic means for the prevention of AKI to safely overcome the persistent barrier of nephrotoxicity during medical intervention.

Deconvoluting hepatic processing of carbon nanotubes.

Single-wall carbon nanotubes present unique opportunities for drug delivery, but have not advanced into the clinic. Differential nanotube accretion and clearance from critical organs have been observed, but the mechanism not fully elucidated. The liver has a complex cellular composition that regulates a range of metabolic functions and coincidently accumulates most particulate drugs. Here we provide the unexpected details of hepatic processing of covalently functionalized nanotubes including receptor-mediated endocytosis, cellular trafficking and biliary elimination. Ammonium-functionalized fibrillar nanocarbon is found to preferentially localize in the fenestrated sinusoidal endothelium of the liver but not resident macrophages. Stabilin receptors mediate the endocytic clearance of nanotubes. Biocompatibility is evidenced by the absence of cell death and no immune cell infiltration. Towards clinical application of this platform, nanotubes were evaluated for the first time in non-human primates. The pharmacologic profile in cynomolgus monkeys is equivalent to what was reported in mice and suggests that nanotubes should behave similarly in humans.

Imaging Tunneling Membrane Tubes Elucidates Cell Communication in Tumors

Intercellular communication is a vital yet underdeveloped aspect of cancer pathobiology. This Opinion article reviews the importance and challenges of microscopic imaging of tunneling nanotubes (TNTs) in the complex tumor microenvironment. The use of advanced microscopy to characterize TNTs in vitro and ex vivo, and related extensions called tumor microtubes (TMs) reported in gliomas in vivo, has propelled this field forward. This topic is important because the identification of TNTs and TMs fills the gap in our knowledge of how cancer cells communicate at long range in vivo, inducing intratumor heterogeneity and resistance to treatment. Here we discuss the concept that TNTs/TMs fill an important niche in the ever-changing microenvironment and the role of advanced microscopic imaging to elucidate that niche.

Abstract LB-095: Distribution and clearance of single-walled carbon nanotubes in mouse tissues: in situ detection, imaging and analysis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Single-walled carbon nanotubes (SWCNT) are the subject of expanding research in the fields of targeted drug delivery and biosensors for disease treatment and monitoring. Functionalization of the SWCNT has proven to enhance the efficiency of distribution in the organism. The goal of this project was to establish and standardize methods of detection and characterization of the local tissue distribution of functionalized SWCNT in mouse models. We investigated the distribution and clearance of SWCNT in several tissues at various times after injection, up to one month. We also try to standardize methods of tracking SWCNT in both live and fixed tissues and image analysis protocols to quantify the distribution of the SWCNT in tissues. Another aim is to evaluate local immune response to SWCNT. We have analyzed liver, kidney, spleen, lung, brain, ovary, colon and small intestine from mice sacrificed 24 hours, 3 days, 7 days and 30 days following the injection of SWCNT. Our evaluation revealed that some organs, like kidney and small intestine, retain SWCNTs for at least a month. Slow rates of SWCNT removal from these organs do not appear to affect the overall health of the animals. We have made multiple attempts to image anesthetized live animals and detect SWCNTs in situ, however, the fluorescent signal emitted from SWCNT is too low to be reliably detected. The tissues must be fixed and signals amplified through immunodetection. Detection of immune markers, especially in immunologically active tissues such as spleen is a challenge for histological experiments, but is extremely important and such experiments is underway. Our findings of SWCNT persistence in certain organs 30 days post-injection is surprising and should be studied further. This observation opens further questions about the effect of long-term presence of SWCNT in some tissues and shows that careful investigation into the advantages and disadvantages of SWCNT retention is necessary. Citation Format: Afsar Barlas, Ke Xu, Yevgeniy Romin, Simone Alidori, Dmitry Yarilin, Ning Fan, Mesruh Turkekul, Sho Fujisawa, David A. Scheinberg, Michael R. McDevitt, Katia Manova-Todorova. Distribution and clearance of single-walled carbon nanotubes in mouse tissues: in situ detection, imaging and analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-095. doi:10.1158/1538-7445.AM2015-LB-095