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. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Toxicol In Vitro. 2021 Feb 27;73:105126. doi: 10.1016/j.tiv.2021.105126

Osteopontin mRNA Expression by Rat Mesothelial Cells Exposed to Multi-Walled Carbon Nanotubes as a Potential Biomarker of Chronic Neoplastic Transformation In vitro

Sreepradha Sridharan 1, Alexia Taylor-Just 1, James C Bonner 1,*
PMCID: PMC8085121  NIHMSID: NIHMS1683121  PMID: 33652123

Abstract

Mesothelioma is a cancer of the lung pleura primarily associated with inhalation of asbestos fibers. Multi-walled carbon nanotubes (MWCNTs) are engineered nanomaterials that pose a potential risk for mesothelioma due to properties that are similar to asbestos. Inhaled MWCNTs migrate to the pleura in rodents and some types cause mesothelioma. Like asbestos, there is a diversity of MWCNT types. We investigated the neoplastic potential of tangled (tMWCNT) versus rigid (rMWCNT) after chronic exposure using serial passages of rat mesothelial cells in vitro. Normal rat mesothelial (NRM2) cells were exposed to tMWCNTs or rMWCNTs for 45 weeks over 85 passages to determine if exposure resulted in transformation to a neoplastic phenotype. Rat mesothelioma (ME1) cells were used as a positive control. Osteopontin (OPN) mRNA was assayed as a biomarker of transformation by real time quantitative polymerase chain reaction (qPCR) and transformation was determined by a cell invasion assay. Exposure to rMWCNTs, but not tMWCNTs, resulted in transformation of NRM2 cells into an invasive phenotype that was similar to ME1 cells. Moreover, exposure of NRM2 cells to rMWCNTs increased OPN mRNA that correlated with cellular transformation. These data suggest that OPN is a potential biomarker that should be further investigated to screen the carcinogenicity of MWCNTs in vitro.

Keywords: carbon nanotubes, osteopontin, mesothelial cells, mesothelioma

Graphical Abstract

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Introduction

Mesothelioma is a rare and fatal cancer of the mesothelial lining surrounding the lungs (i.e. pleura) caused by abnormal division of the mesothelial layer which leads to tumor formation. Approximately 3,000 new cases of mesothelioma are diagnosed each year in the U.S. (Robinson, 2012). In 80% of patients with mesothelioma, exposure to asbestos occurs about thirty years prior to initial symptoms (Jaurand and Fleury-Feith, 2005). Patients usually survive less than a year after diagnosis (Sekido, 2008). Mesothelioma is primarily associated with asbestos exposure and certain types of asbestos fibers possess greater carcinogenicity than others (Ray and Kindler, 2009; Tomasetti et al., 2009). For example, crocidolite “blue” asbestos is a rigid, rod-like fiber that is linked to a high incidence of mesothelioma in the human population as compared with chrysotile “white” asbestos, which is a curly or tangled, flexible fiber (Mossman et al., 2011). Rats serve as a useful animal model for asbestos-induced mesothelioma. Rigid, rod-like asbestos can directly interact with mesothelial target cells to cause cell membrane disruption, DNA damage and release of cytokines that play important roles in the pathogenesis of mesothelioma (Broaddus et al., 2011). As a result, normal mesothelial cells may be induced to proliferate or sustain genetic alterations leading to cellular transformation and tumor development.

The emergence of nanotechnology has generated engineered nanomaterials termed multiwalled carbon nanotubes (MWCNTs) that are structurally similar to asbestos. MWCNTs have superior conductivity and high-tensile strength and are increasingly used in electronics and structural materials (Pagona and Tagmatarchis, 2006). Like asbestos fibers, long, rigid MWCNTs have a high length to width aspect ratio, which leads to frustrated phagocytosis by macrophages, leading to generation of reactive oxygen species (ROS) and decreasing clearance of MWCNTs from the lungs (Boyles et al., 2015). Similar to asbestos, there is an array of different MWCNT types. For example, a rigid, rod-like MWCNTs (e.g., Mitsui-7) possess carcinogenicity in rodents and have been classified by the International Agency for Research on Cancer as a class 2B probable carcinogen in humans (Grosse et al., 2014; Nagai et al., 2011). Numerous rodent studies indicate that MWCNTs are biopersistent and cause lung disease (inflammation, fibrosis, cancer) after inhalation and therefore could have adverse health effects in humans if inhaled (Huizar et al., 2011; Poland et al., 2008; Sargent et al., 2014; Suzui et al., 2016; Takagi et al., 2008; Wang et al., 2014). Our lab previously showed that carbon nanotubes delivered to the lungs of mice or rats by inhalation or aspiration cause inflammation and fibrosis within the pulmonary interstitium and surrounding airways (Cesta et al., 2010; Ryman-Rasmussen et al., 2009a; Taylor et al., 2014). In addition to causing disease within the pulmonary tissue, we have demonstrated that MWCNTs inhaled by mice also reach the pleura that surrounds the lungs and persist for months and interact directly with mesothelial cells (Ryman-Rasmussen et al., 2009b). Others have reported that mice exposed intratracheally to MWCNTs develop lung cancer and mesothelioma (Sargent et al., 2014;Suzui et al., 2016). However, it is unknown whether MWCNTs cause mesothelioma in humans since clinical signs of this cancer take decades to manifest and human exposures to MWCNTs have occurred mainly over the past couple of decades.

The pleural lining surrounding the lungs is composed of a monolayer of mesothelial cells. Chronic exposure of human mesothelial cells to asbestos in vitro causes cellular transformation that can be quantified by loss of contact inhibition, formation of multilayered cellular foci, invasion, migration through Matrigel-coated membranes, colony formation on soft agar and tumor sphere formation (Cleaver et al., 2014, Lohcharoenkal et al., 2013; Qi et al., 2013). A normal rat pleural mesothelial (NRM2) cell line and a rat mesothelioma (ME1) cell line (derived from spontaneous mesothelioma) have been isolated and cultures can be stably maintained for months over multiple sub-passages in vitro (Bermudez et al., 1990; Rutten et al., 1995). NRM2 cells exhibit an indefinite proliferative capacity in culture, exhibit a typical cobblestone appearance when confluent, and their growth in culture is contact inhibited by neighboring cells to form a monolayer. In contrast, ME1 cells exhibit loss of contact inhibition in cell culture and loss of monolayer integrity. Importantly, ME1 cells but not NRM2 cells cause tumor promotion when injected into immunosuppressed nude mice (Funaki et al., 1991).

Osteopontin (OPN) is a glycoprotein often overexpressed in lung and ovarian cancer in humans (Wei et al., 2017). OPN plays a role in the regulation of cell-matrix interactions and cell signaling (Pagel et al., 2013). An increase in OPN expression often correlates with tumor invasion and metastasis (Pass et al., 2005). In patients with exposure to asbestos versus patients with no exposure to asbestos, there is a significant increase in serum OPN expression (Pass et al., 2005). Moreover, OPN has been shown to be overexpressed in serum of patients with mesothelioma (Amany et al., 2013).

The aim of this study is to determine the relative potential for tangled (t)MWCNTs versus rigid (r)MWCNTs to cause neoplastic transformation of normal rat mesothelial cells after chronic exposure and serial sub-passages in vitro. We hypothesized that increased OPN expression in vitro would correlate with greater invasive properties in rat NRM2 cells exposed to MWCNTs. To our knowledge, this is the first time OPN has been investigated as an indicator of neoplastic transformation in mesothelial cells by carbon nanotubes.

Materials & Methods

Nanomaterials.

Tangled (t)MWCNTs were obtained from Helix Materials Solutions Inc. (Richardson, TX). Rigid (r)MWCNTs, also known as Mitsui-7, were manufactured by Mitsui & Co. (Tokyo, Japan) but were obtained from NIOSH. Various physicochemical characteristics of tMWCNTs and rMWCNTs including rigidity, diameter, length, surface area, zeta potential, and residual trace metal content have been previously determined by our lab and others (Duke et al., 2017; Hilton et al., 2017; Porter et al., 2010; Ryman-Rasmussen et al., 2009b). These physicochemical characteristics are summarized in Supplemental Table 1. MWCNTs were suspended in 0.1% pluronic solution (Sigma, St. Louis, MO) diluted in Dulbecco’s phosphate-buffered saline (DPBS) to a concentration of 0.1 μg/ml.

Cell Culture.

Normal rat mesothelial (NRM2) cells and rat mesothelioma (ME1) cells were obtained from Edilberto Bermudez at the Hamner Institutes for Health Sciences (Research Triangle Park, NC). NRM2 cells were isolated from the parietal pleural mesothelium of a male Fischer-344 rat (Bermudez et al., 1990). ME1 cells were isolated from spontaneous mesothelioma tumors on the peritoneum of a male Fisher-344 rat (Funaki et al., 1991). Cells were grown in 6-well cell culture plates in a 1:1 mixture of Kaign’s (F12K) medium and Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA), supplemented with 10% fetal bovine serum (FBS) (Genesee Scientific, Morrisville, NC). NRM2 cells were exposed to tMWCNT or rMWCNTs for 45 weeks. Cells were passaged twice a week until passage 85 was reached. At each passage, MWCNTs (0.1 μg/ml) were re-introduced to the cell medium for a final MWCNT concentration of 0.02 μg/cm2 per well. The dosing strategy and concentration of MWCNTs were based on previous studies using human mesothelial cells or human airway epithelial cells (Lohcharoenkal et al., 2013; Stueckle et al., 2017a,2017b).

Cell imaging.

At passage 12 and 80, the NRM2 cells were grown on glass coverslips in 6-well cell culture plates for 24 hours and stained with the Diff-quik stain set (Siemens, Newark, DE). Photomicrographs were taken at 100x magnification with the Olympus light microscope BX41 (Center Valley, PA). ME1 cells were imaged at passage 13 for comparison as a positive control.

OPN mRNA Expression.

During the 45-week experiment, mRNA was collected once a week from the NRM2 cells and only one time from the ME1 cells (passage 12). Expression of the mRNA encoding osteopontin (OPN) was measured using real-time qPCR. The mRNA was collected from cells using the Quick-RNA MiniPrep kit (Zymo Research, Irvine, CA) according to the manufacturer’s protocol. The isolated mRNA was used to create cDNA using the High Capacity cDNA Reverse Transcription Kit (ThermoFisher, Waltham, MA). The FastStart Universal Probe Master (Rox) (Sigma, St. Louis, MO) was used to determine the comparative Ct fold change in mRNA expression of OPN normalized with β2-microglobulin (B2M) as the endogenous control on the StepOnePlus real-time qPCR System (ThermoFisher, Waltham, MA).

Invasion Assay.

The CultureCoat® Medium BME Cell Invasion Assay (R&D Systems, Minneapolis, MN) was used according to the manufacturer’s protocol to analyze the ability of cells to exhibit neoplastic characteristics. Low passage NRM2 cells (P12) were used as a negative control and ME1 cells (P13) were used as a positive control in comparison to the high passage NRM2 cells exposed to MWCNTs (P85). Each experiment contained 16 technical replicates per treatment group in 96-well plates.

Statistics.

Statistical analysis of the data was performed using GraphPad Prism version 5.0 (GraphPad Software Inc.). A paired Student’s t-test was used to determine significance between samples. A two-way ANOVA was used with a Bonferroni post-test to compare between multiple treatments.

Results

TEM imaging of the MWCNTs.

Transmission electron microscopy was used to characterize the morphometry of the tMWCNTs and rMWCNTs used in this study (Fig 1). These MWCNTs have been previously characterized to determine average length, width, residual metal content and rigidity (See Supplemental Table 1). We previously determined that rMWCNTs are 7-fold more rigid than the tMWCNTs (Duke et al., 2017).

Fig. 1.

Fig. 1.

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Transmission electron microscope images of nanotubes used in our in vitro experiments. A) tangled (tMWCNT). B) rigid (rMWCNT).

Chronic exposure of normal mesothelial cells to rMWCNTs results in loss of contact inhibition similar to mesothelioma cells.

Cultured normal rat mesothelial cells (NRM2) were grown on glass slides for 24 hours, fixed and stained with Diff-Quick to assess cell morphometry. Early passage normal NRM2 cells (P12) formed a cobblestone monolayer (Fig. 2). Late passage NRM2 cells (P80) that were not treated with MWCNTs also formed a confluent monolayer, although cells were more densely packed compared to early passage NRM2 showing less contact inhibition. Late passage P80 NRM2 cells chronically treated with tMWCNT maintained a dense monolayer, while treatment with rMWCNT resulted in altered cell morphometry that included optically dense foci of clusters of cells. Transformed ME1 cells, that were used as a positive control, also exhibited optically dense foci of cell clusters that indicated piling up, loss of contact inhibition, and loss of monolayer integrity.

Fig. 2.

Fig. 2.

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Light microscopy images of NRM2 and ME1 cells at low passages (P12–13) and high passage (P80) with or without exposure to tMWCNTs or rMWCNTs.

Osteopontin (OPN) mRNA expression and cell invasion in NRM2 and ME1 cells as indicators of mesothelioma.

We first compared OPN mRNA expression by NRM2 and ME1 cells that were not treated with MWCNTs. The cells were grown to confluence in 6-well plates in 10% FBS-DMEM and rendered quiescent in serum-free medium for 24hrs before mRNA isolation and collection. The results of qPCR analysis showed that ME1 cells produced significantly more OPN mRNA than NRM2 cells (Fig. 3A). ME1 cells also showed higher invasive properties compared to NRM2 cells, which confirms that they are a reliable positive control for neoplastic potential (Fig. 3B).

Fig. 3.

Fig. 3.

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OPN mRNA expression and cell invasion in NRM2 (P14, P12) & ME1 (P15, P13) cells as indicators of mesothelioma. A) OPN mRNA measured by real time qPCR. B) Quantification of cell invasion across an extracellular matrix after 24hrs. *** p < 0.001, ** p < 0.01 compared to control. Data are from a single experiment representative of three independent experiments.

Osteopontin mRNA expression increases with chronic exposure to rMWCNTs.

OPN mRNA expression was measured in NRM2 cells chronically treated with either tMWCNTs or rMWCNTs at passages 42, 46, 51, 55, 58, and 61. NRM2 cells not treated with MWCNTs were used as a negative control. The expression of OPN mRNA in NRM2 cells treated with rMWCNTs, but not tMWCNTs, was significantly higher than in NRM2 cells treated with control media alone, although OPN mRNA expression was variable among passages (Fig. 4). In addition, OPN mRNA in NRM2 cells treated with rMWCNTs was significantly higher than in NRM2 cells treated with tMWCNTs at passages 42, 46, 51, and 61, but not at passages 55 and 58 (Fig. 4).

Fig. 4.

Fig. 4.

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Osteopontin mRNA expression with exposure to tMWCNTs or rMWCNTs with increasing cell passages. ***p < 0.001, *p < 0.05 compared to passage # control. ###p < 0.001, ##p < 0.001 between tMWCNT and rMWCNT. Data are from a single experiment representative of three independent experiments.

High passage NRM2 cells chronically exposed to tMWCNTs or rMWCNTs are more invasive.

Invasion assays were performed to assess the neoplastic properties of late passage (P85) NRM2 cells chronically exposed to tMWCNTs and rMWCNTs. Late passage (P85) NRM2 cells not exposed to MWCNTs had a similar number of invading cells compared to low passage (P12) NRM2 cells. Both MWCNT types increased the number of invasive cells when compared to the late passage control cells (Fig. 5). However, NRM2 cells exposed to rMWCNTs had a higher number of invading cells compared to cells exposed to tMWCNTs (Fig. 5).

Fig. 5.

Fig. 5.

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Cell invasion assay with high passage NRM2 cells exposed to tMWCNTs or rMWCNTs. Low passage (P12) as negative control. *** p < 0.001, ** p < 0.01 compared to P85 control. ###p < 0.001 between P85 tMWCNT and P85 rMWCNT. Data are from a single experiment representative of three experiments. Each experiment contained 16 replicates per group.

Discussion

In this study, we show that chronic exposure of normal rat mesothelial (NRM2) cells in vitro to MWCNTs induces the transformation to an invasive phenotype that is similar to a rat mesothelioma cell line (ME1). Moreover, we demonstrated a differential response of NRM2 cells to two different types of MWCNTs with contrasting characteristics. NRM2 cells chronically exposed to rod-like, rigid rMWCNTs had higher OPN mRNA expression and exhibited greater invasive properties compared to NRM2 cells chronically exposed to tangled tMWCNTs. NRM2 cells also exhibited changes in morphology after chronic exposure to MWCNTs. Untreated NRM2 cells formed a cobblestone monolayer, while NRM2 cells treated chronically with rMWCNTs exhibited a morphology similar to that observed with ME1 cells, which displayed piling behavior and loss of contact inhibition. ME1 cells also showed higher invasive properties and higher OPN mRNA expression compared to NRM2 cells.

Other studies support the concept that long, rigid MWCNT cause cell transformation. Two studies by Stueckle et al., (2017a and 2017b) using small airway epithelial cells demonstrated a greater proliferative capacity after prolonged exposure to MWCNTs. The study by Stueckle et al., 2017a used Mitsui-7 (the same rMWCNT used in our study) as a positive tumor promoter nanomaterial for two other nanoparticles, CeO2 and Fe2O3, while the companion study by Stueckle et al., 2017b used tangled MWCNTs and functionalized derivatives. They concluded that Mitsui-7 were more potent at cell transformation than all other tangled MWCNTs tested (Stueckle et al., 2017a, 2017b). These observations are consistent with our findings that rMWCNTs are much more potent than tMWCNTs in stimulating NRM2 cells for greater OPN mRNA expression and increased invasive activity. Another study by Polimeni et al., 2016 illustrated that long-term exposure to rigid MWCNTs (Mitsui-7) in vitro caused epithelial-mesenchymal transformation (EMT) in human bronchial epithelial cells. These cells exhibited greater “disorganization” with decreased contact inhibition and altered cell morphology. EMT was triggered by long term exposure to rMWCNTs by stimulating TGF-β1 secretion, activating Akt and inhibiting GSK-3β. (Polimeni et al., 2016). Finally, a recent study by Reamon-Buettner et al., 2020 showed that primary human mesothelial cells undergo premature cellular senescense after treatment with long, rigid MWCNTs and this could be related to carcinogenesis.

OPN has been reported as a biomarker for mesothelioma in humans exposed to asbestos. Patients with pleural mesothelioma were found to have significantly higher serum OPN levels compared to patients with no exposure to asbestos (Pass et al., 2005). A longer duration of exposure to asbestos also correlated with higher levels of detectable serum OPN. Additionally, higher tissue levels of OPN correlated with decreased survival in patients with mesothelioma. This study also found that ELISA assays for OPN are potentially useful for detecting stage I mesothelioma in high-risk patients. OPN has also been determined to be an accurate diagnostic indicator of malignant pleural mesothelioma in patients (Amany et al., 2013). The study by Grigoriu et al., 2007 also supports the role of OPN as a diagnostic marker. Although this study found that the detection of serum OPN in patients was beneficial in diagnosing mesothelioma, the detection of pleural OPN did not serve as well to differentiate mesothelioma patients from those with benign pleural lesions (Grigoriu et al., 2007). Therefore, OPN alone is not a perfect biomarker and a diagnostic panel of biomarkers that includes OPN would likely improve the diagnosis of mesothelioma and the value of in vitro assays for predicting the risk of mesothelioma caused by inhaled fibers, including asbestos and MWCNTs.

While our findings suggest that OPN could be a potential biomarker for the neoplastic-like transformation of rat pleural mesothelial cells exposed to rMWCNTs, further investigation is required to determine whether OPN would be useful for predicting mesothelioma in humans exposed to MWCNTs of any kind. Indeed, it has yet to be shown that MWCNTs cause mesothelioma in humans. However, based on studies in rodents, it seems likely that at least some types of MWCNTs will be a risk for mesothelioma and lung cancer in people (Poland et al., 2008; Sargent et al., 2014; Sakamoto et al., 2018; Suzui et al., 2016; Takagi et al., 2012). Given that occupational exposure to MWCNTs has occurred during the relatively recent emergence of nanotechnology over the past couple of decades (Fatkhutdinova et al., 2016), it is possible that mesothelioma has not yet been in documented in humans due to the long latency period of the disease, which can take decades before clinical symptoms are seen (Mossman et al., 2011).

An abundance of evidence from rodent studies suggests that exposure to rigid, rod-like MWCNTs could cause mesothelioma in humans. One particular study by Sakamoto et al., 2018 tested the carcinogenicity of 7 different types of MWCNTs, 4 being more rigid and 3 tangled. They found that all of the rigid MWCNTs induced mesothelioma after 52 weeks when administered via intraperitoneal injection to rats. In comparison, the tangled MWCNTs had a tumor induction rate of only ~6% (Sakamoto et al., 2018). Huizar et al., 2011 illustrated that oropharyngeal instillation of MWCNTs with a tangled morphology in mice resulted in a higher occurrence of a granulomatous inflammatory response, but there was no mention of mesothelioma in this study. There was also an increased mRNA expression of OPN in bronchoalveolar lavage cells 60 to 90 days post-exposure to MWCNTs (Huizar et al., 2011). Similarly, Chernova et al., 2017 found that the instillation of long, straight MWCNTs into the pleural cavity of mice increased inflammatory lesions in the pleura. This study concluded that the neoplastic transformation of mesothelial cells exposed to MWCNTs occurred due to the deletion and silencing of key tumor suppressor genes (Chernova et al., 2017). Takagi et al., 2012 showed that a year post-exposure to an intraperitoneal injection of rigid (Mitsui-7) MWCNTs in mice led to mesothelioma with dose determining the severity (Takagi et al., 2012). Finally, Suzui et al., 2016 studied the effects of intratracheal installation of rigid MWCNTs in rats. They found that MWCNTs were localized to lung alveoli, the mediastinal space, and lymph nodes. The occurrence of mesothelioma was higher than in the control groups and they concluded that the instillation of MWCNTs in lung tissue has the potential to cause malignant mesothelioma (Suzui et al., 2016).

The findings detailed in our study suggest that OPN could be a potential biomarker for neoplastic-like transformation of normal rat mesothelial (NRM2) cells chronically exposed to MWCNTs. Chronic exposure to rigid (r)MWCNTs resulted in cellular transformation into a more invasive, neoplastic-like phenotype while also increasing OPN mRNA expression, when compared to tangled (t)MWCNTs. Increased invasion and OPN mRNA expression were also characteristics of rat mesothelioma (ME1) cells. Based on this, our results suggest that rMWCNTs could have a similar neoplastic potential as asbestos in humans and exposure could lead to mesothelioma, whereas tMWCNTs appear to have low potential to transform mesothelial cells and increase OPN mRNA expression. A caveat of our study is that only certain passages of NRM2 cells exposed to rMWCNTs (P42, P46, P51, P61) displayed significantly increased OPN mRNA levels compared to control or tMWCNT-treated NRM2 cells, while two other passages (P55 and P58) did not. P85 cells used for invasion assays were not assayed for OPN mRNA. The reason for variability in OPN mRNA levels among passages is not known. Because the serial passage experiment was conducted over 45 weeks (~1 year), it was not feasible to further investigate passage variability. In order to more thoroughly conclude that rMWCNTs cause neoplastic transformation of pleural mesothelial cells, further investigation should determine whether chronic exposure to rMWCNTs causes colony formation in soft agar in vitro and tumor promotion in vivo when injected into immunosuppressed nude mice. Also, our observations of neoplastic-like transformation by rMWCNTs are based on a single rat mesothelial cell line, so future work should evaluate whether our findings are extendable to other normal mesothelial cells. Nevertheless, the findings reported herein indicate that the NRM2 cell culture model is a useful in vitro alternative to rodent testing for predicting neoplastic transformation and risk for mesothelioma induced by chronic exposure to different types of MWCNTs.

Supplementary Material

1

Supplemental Table 1. Comparison of physicochemical characterization of bulk tangled (t)MWCNT and rigid (r)MWCNT. ND, not determined.

Highlights.

  • Two types of multiwalled carbon nanotubes (tangled or rigid) were tested for their potential to cause neoplastic transformation of normal rat pleural mesothelial cells in vitro.

  • Rigid carbon nanotubes, but not tangled ones, caused loss of cell contact inhibition and increased cell migration in an invasion assay after 85 serial passages over 45 weeks.

  • Osteopontin mRNA expression was significantly increased by mesothelial cells over certain passages by rigid carbon nanotubes, but not tangled carbon nanotubes.

  • These data suggest that OPN is a potential biomarker that should be further investigated for screening the neoplastic potential of MWCNTs in vitro.

Acknowledgements

This work was supported by National Institute of Environmental Health (NIEHS) grant P30ES025128, NIEHS grant R01ES020897 and National Science Foundation (NSF) grant CBET-1530505.

Footnotes

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Declaration of competing interests

The authors declare that they have no known competing financial interests or relationships that could have appeared to influence the work reported in this paper.

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Supplementary Materials

1

Supplemental Table 1. Comparison of physicochemical characterization of bulk tangled (t)MWCNT and rigid (r)MWCNT. ND, not determined.

NIHMS1683121-supplement-1.docx (16KB, docx)

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