Exosomes from Irradiated Nonsmall Cell Lung Cancer Cells Reduced

7 days ago - Department of Respiration, Peking University Shenzhen Hospital, Lianhua Road N0.1120, Shenzhen , 518036 Guangdong Province , China. § De...
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Exosomes from irradiated non–small cell lung cancer cells reduced sensitivity of recipient cells to anaplastic lymphoma kinase inhibitors Hao Wu, Chao Zeng, Yiwang Ye, Jixian Liu, Zhimin Mu, Yuancai Xie, Baokun Chen, Qiaohong Nong, and Da Wu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00059 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 30, 2018

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Molecular Pharmaceutics

Exosomes from irradiated non–small cell lung cancer cells reduced sensitivity of recipient cells to anaplastic lymphoma kinase inhibitors Hao Wu1, Chao Zeng2, Yiwang Ye1, Jixian Liu1, Zhimin Mu1, Yuancai Xie1, Baokun Chen1, Qiaohong Nong3*, Da Wu1*

1,

Department of Thoracic Surgery, Peking University Shenzhen Hospital, Lianhua Road

N0.1120, Shenzhen 518036, Guangdong Province, China 2,

Department of Respiration, Peking University Shenzhen Hospital, Lianhua Road N0.1120,

Shenzhen 518036, Guangdong Province, China 3,

Department of Oncology, Peking University Shenzhen Hospital, Lianhua Road N0.1120,

Shenzhen 518036, Guangdong Province, China

*Corresponding author: Da Wu, Department of Thoracic Surgery, Peking University Shenzhen Hospital, Lianhua Road N0.1120, Shenzhen 518036, Guangdong Province, China E-mail: [email protected]

*Co-corresponding author: Qiaohong Nong, Department of Oncology, Peking University Shenzhen Hospital, Lianhua Road N0.1120, Shenzhen 518036, Guangdong Province, China E-mail: [email protected]

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Abstract Exosomes, released from various cell types, serve as vehicles of intercellular communication. Rearranged anaplastic lymphoma kinase (ALK) has been detected in exosomes released from cancer cells in ALK-positive non-small cell lung cancer (NSCLC), however, the functional consequence of ALK in exosomes has not been studied. This study aims to address whether exosomal ALK release is affected by stress, and whether exosomal ALK can modulate survival of recipient cells in vitro and in vivo. Exosomes, isolated from ALK-containing H3122 cells with (Exo-Apo) or without (Exo-Ctrl) irradiation treatment, were transferred to recipient H3122 cells in vitro or mouse xenograft in vivo. Western blot, flow cytometry, MTT, and xenograft were employed to respectively assess activation of ALK pathway, apoptosis, cell viability, and tumor growth. Exo-Apo contained much higher levels of phosphorylated ALK (pALK) than Exo-Ctrl, and activated AKT, STAT3 and ERK pathway in recipient H3122 cells. ALK specific inhibitors, including Crizotinib, Ceritinib, and TAE684, exhibited less effects on H3122 cells pre-incubated with Exo-Apo than on those treated with Exo-Ctrl in either inhibition of cell viability or promotion of apoptosis. Moreover, in an H3122 xenograft model, Exo-Apo treatment resulted in a greater tumor growth and less sensitivity to Ceritinib than Exo-Ctrl treatment. ALK protein cargo in exosomes could be a key element to drive tumor growth and compromise therapeutic efficacy of ALK inhibitors for ALK-positive NSCLC.

Keywords: Exosomes; non-small cell lung cancer; anaplastic lymphoma kinase

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Introduction Rearrangement in anaplastic lymphoma kinase (ALK) gene, an oncogene found in 2-7% of non-small cell lung cancer (NSCLC), defines a distinct molecular subtype of lung cancer (1). Constitutive activation of ALK by rearrangements of ALK gene results in tumorigenesis through activation of downstream signaling targets, including AKT, signal transducer and activator of transcription 3 (STAT3), and extracellular regulated kinase (ERK) (2). Rearranged ALKs have been detected in platelets, urine, and exosomes released by tumor cells in NSCLC patients (3, 4). ALK in liquid biopsies is not only putatively regarded as a biomarker for the progress of lung cancer during treatment, but also may guide the choice of therapeutic strategies. Exosomes are a subclass of extracellular micro-vesicles that are shed directly from the plasma membrane, and are released into extracellular environment by different cell types in both physiological and pathological conditions, including tumor cells (5, 6). Exosomes and their composition mirror the parental cells, thus, serve as vehicles of intercellular communication for transferring proteins, lipids, and RNAs (5, 6). In this sense, it has been reported that exosomes provide a means carrying information about the arrival of cancer cells or exosomal cargos from other sites, and creating special conditions in oncogenic reprogramming, tumor progression, metastasis, and resistance to chemotherapy (6-8). The exosomal composition shows dynamic alterations in the presence of cellular stressors, such as chemotherapy, irradiation, or oxidative stress, and subsequently modifies the communication of exosomes with surrounding recipient cells (9-11). In the present study, irradiation elevated phosphorylation of ALK in exosomes released from NSCLC cell line (H3122). Exosomes carrying p-ALK activated ALK pathways, promoted cell survival, and led to resistance to ALK inhibitors in recipient cells in vitro and in vivo. The

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results support the notion that exosomes from NSCLC carry rearranged ALK and may be emerging players in tumor growth and drug resistance.

Experimental Section Cell culture and reagents H3122 (purchased from Hede Biotechnology LTD, Beijing, China) and PC9 cells (purchased from Shengbo Biotechnology LTD, Shanghai, China) were maintained under a humidified atmosphere of 5% CO2 at 37 oC in RPMI 1640 medium (Sigma, St Louis, MO, USA), supplemented with 10% fetal bovine serum (Gibco, USA), 100 U/ml streptomycin (Thermo Fisher Scientific), and 1 mM sodium pyruvate (Sigma, USA). H3122 and PC9 cells were exposed to irradiation of γ-rays (0.45 Gy per min) at doses of 3, 6, and 9 Gy from a 137cesium source (HWM-D2000, Wälischmiller Engineering). . All drugs were dissolved in dimethyl sulfoxide (DMSO), stored at −70 oC, and diluted in fresh media just before experiments. Isolation of exosomes We isolated exosomes from culture supernatants by a series of centrifugation procedures as previously described (10). Briefly, cells were plated in 10 cm dishes, and exosome-depleted FCS (edFCS) medium was added prior to irradiation. To obtain edFCS, high-speed centrifugation (100,000 g) was performed at 4 oC for 14 hours to remove bovine exosomes from fetal calf serumand. The culture medium was collected 24 hours later, and was centrifuged at 10,000 g for 30 minutes at 4 oC. The supernatant was retained and passed through a filter (0.22 µm), then, was further centrifuged at a higher speed (100,000 g) for 75 min at 4 oC. Phosphate buffer solution (PBS) was used to suspend the exosome pellet in the precipitates. A second round

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Molecular Pharmaceutics

ultracentrifugation (100,000 g, 75 minutes, 4 oC) was applied. Finally, the exosome pellet was resuspended in fresh PBS and were stored in a freezer (−20 oC). For the biological assay of exosomes, we incubated recipient cells in edFCS with exosomes isolated from donor cells with and without exposure to irradiation. Immunoblot analysis Cells were harvested using cell scrapers. Protein was extracted with RIPA buffer and was stored at −20 °C. Western blot assay was performed with a standard protocol to detect designated proteins in samples containing either 10 µg cellular protein or 10 µl exosome lysate. Polyclonal antibodies of human phosphorylated ALK (pY1604) (p-ALK), ALK, phosphorylated signal transducer and activator of transcription 3 (STAT3) (p-STAT3), STAT3, phosphorylated AKT (p-AKT), AKT, extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), Alix, CD63, GM130, and tubulin were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary horseradish peroxidase-conjugated antibodies and PierceTM western blotting substrate (Thermo Fisher Scientific, Shanghai, China) were used for visualization and semiquantification of the corresponding proteins. Cell viability assay Cells were seeded in 96-well plates and were treated with concentrations of ALK inhibitors (1-1,000 nM) for 48 hours. Growth inhibition was assessed by MTT assay as described previously using MTT assay kit (Abcam, the US) (12). The curves were fitted using a non-linear regression model to depict a sigmoidal dose-response relationship. Apoptotic changes in the plasma membrane Cells, seeded in 10 cm plates, were treated with Ceritinib, Crizotinib, TAE684, or Erlotinib (purchased from Selleck Chemicals). Apoptosis was evaluated with the flow cytometry (BD

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Bioscience, the US) after cells were stained with a FITC Annexin V/Dead Cell Apoptosis Kit with PI (Thermo Fisher Scientific, China) (13). The conjugated green (Annexin) and red (PI) fluorescence were respectively excited, and the emission lights were collected. The cells stained with Annexin or PI represent apoptotic or necrotic cells, which were automatically counted by the equipment. Xenograft assay Nude mice (nu/nu; 6–8 weeks old) were used for in vivo studies. The care and use for these mice comply with a protocol approved by the Animal Care and Use Committee of Peking University Shenzhen Hospital. A suspension of H3122 cells (5 × 106 in 0.2 ml of PBS) was inoculated subcutaneously into the lower-right quadrant of the flank of each mouse under 2% Isoflurane anesthesia. Mice carrying tumor with a volume not less than 150 mm3 were randomly allotted to four groups (n=15 per group). Ceritinib (10 mg/kg) or vehicle was daily administrated by oral gavage. Tumors were measured twice per week using calipers, and volume was calculated using the formula (length × width2 × 0.52). Mice were monitored daily for body weight and general conditions. Statistical analysis Quantitative data were presented as mean ± S.E.M. and were analyzed with the unpairedtwo-tailed Student’s t-test or one-way ANOVA as needed. A p value less than 0.05 was considered statistically significant.

Results Elevation of apoptotic markers and ALK phosphorylation in exosomes from irradiationtreated H3122 cells

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To evaluate whether ALK is presented in exosomes released by H3122 cells, and whether cellular stress, such as apoptosis, can change ALK in exosomes, we first used irradiation as cellular stressor, and examined the effects of irradiation on apoptosis marker proteins: caspase 3 and Bax, and an anti-apoptotic marker protein: Bcl-2. Treatment of irradiation (3 Gy or 6 Gy) elevated the levels of caspase 3 and Bax, but, reduced the level of Bcl-2 in H3122 lysates (Fig.1A, B). Next, exosomes were isolated from the culture medium of irradiated (3 Gy and 6 Gy) or non-irradiated H3122 cells by differential ultracentrifugation. Exosomes purified from either irradiated (3, 6 Gy) (Exo-Apo) or non-irradiated (0 Gy) cells (Exo-Ctrl) showed the expected enrichment of the exosome marker proteins, ALIX and CD63, relative to cellular lysates (Fig.1C). GM130, a marker protein of Golgi apparatus, is absent in exosomes, and was not detected in our exosome lysate preparations, but was highly abundant in the cellular lysates (Fig. 1C). The data demonstrate that our exosome preparations were not contaminated by intracellular organelles derived from apoptotic bodies or dead cells. As 6 Gy irradiation exhibited stronger inhibition of Bcl-2 than 3 Gy irradiation in H3122 cells (Fig. 1B), we irradiated H3122 cells at this dose to prepare Exo-Apo for the following steps of experiments. Western blotting detected high levels of total ALK, but relatively low levels of phosphorylated (p-) ALK in Exo-Ctrl (0 Gy) (Fig. 1D, E). Irradiation (6 Gy) increased levels of exosomal p-ALK and total ALK by 4-fold and one-fold, respectively (Fig.1D, E). These data suggest that ALK was presented in H3122 exosome preparations; irradiation promotes apoptosis and activates ALK pathway in exosomes. Additionally, we measured ALK and p-ALK levels in lysate from irradiated H3122 cells. We observed that irradiation increased the levels of p-ALK, but not ALK (Fig. 1F, G). The

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magnitude of the increase in lysates was half of that in exosomes (Fig. 1E, G), suggesting that ALK pathway in exosome may be more sensitive to irradiation than that in cytoplasm. Exo-Apo activated AKT, STAT3 and ERK signaling pathways in recipient H3122 cells We were wondering whether upregulated exosomal ALK exhibits biological effects on the recipient cells. To address this question, we isolated exosomes from ALK-positive H3122 cells and ALK-negative PC9 cells with and without irradiation treatment (6 Gy). Then, we added isolated exosomes to recipient cells (H3122), and measured downstream effectors of ALK, including AKT, ERK and STAT3. Adding Exo-Apo (exosomes from irradiated H3122 cells) into culture medium increased levels of p-AKT, p-ERK, and p-STAT3 in recipient cells, compared with adding exosomes from non-irradiation-treated H3122 cells in culture medium (Fig. 2A-C). These processes could rely on irradiation-induced enhancement of ALK-pathway in exosomes, because adding exosomes from ALK-negative PC9 cells with and without irradiation in culture medium of recipient H3122 cells resulted in the same levels of p-ALK, p-AKT, and p-STAT3 in these recipient cells (Fig. 2A-C). These results suggest that irradiation-induced alterations in the ALK pathway accompanied with apoptosis could be transferred to recipient cells via exosomes, and subsequently trigger the downstream signaling cascades of ALK in recipient cells. Exo-Apo conferred resistance of H3122 cells to ALK inhibitors To address whether p-ALK enriched exosomes from irradiation-treated H3122 cells (i.e. Exo-Apo) change responses of recipient H3122 cells to ALK inhibitors, we performed MTT assay to measure viability of recipient cells. Exo-Apo or Exo-Ctrl were added into the culture medium of H3122 recipient cells. Three ALK specific inhibitors, including Crizotinib, Ceritinib, and TAE684, were then used to block ALK activity. Treatment with Crizotinib, Ceritinib, or TAE684 dose-dependently reduced viability of recipient cells with Exo-Apo or Exo-Ctrl in

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culture medium (Fig. 3A-C). The dose-response relationships of these 3 drugs in reducing viability of H3122 cells were similar to previously reported ones (14). Interestingly, all ALK inhibitors displayed higher IC50 for reduction of cell survival in Exo-Apo-treated group than in Exo-Ctrl-treated group (Fig. 3D). These results demonstrate that exosomal ALK released from irradiated tumor cells reduced sensitivity of recipient cells to ALK inhibitors. PC-9 cells lack of ALK signaling pathway, but express epidermal growth factor receptors (EGFR), and EGFR inhibitor, Erlotinib (purchased from Selleck Chemicals), effectively causes death of PC-9 cells (15). To test whether Exo-Apo from H3122 cells changes the sensitivity of recipient PC-9 cells to Erlotinib, we treated the PC-9 cells with either Exo-Apo or Exo-Ctrl from H3122 cells, tested the dose-response relationships of Erlotinib in reducing viability of PC-9 cells. We obtained similar dose-response relationships between these two conditions. Taken together, these results suggest that Exo-Apo-mediated resistance to anti-cancer drugs may have somewhat selectivity to ALK inhibitors, but not to EGFR-inhibitors. Next, the effect of ALK inhibition on apoptosis of recipient cells was compared between Exo-Apo-treated and Exo-Ctrl-treated cells. Fig. 3 shows that Ceritinib was the most potent ALK inhibitor to cause death of H3122 cells. Therefore, we chose Ceritinib to examine apoptosis enhancement following ALK inhibition. For concentration of selection of Ceritinib, we used a dose that had minimal effect on cell survival. When H3122 cells were treated with either ExoApo or Exo-Ctr, we did not find significant difference in apoptosis rate of recipient H3122 cells in these two conditions (left panels in Fig. 4A, and Fig. 4B). As expected, Ceritinib (2 nM for 24 h) significantly increased the number of Annexin V-positive cells in both Exo-Apo and Exo-Ctrl treated H3122 cells, representing the enhancement of apoptosis (Right panels in Fig. 4A, and Fig. 4B). However, the enhancement of apoptosis by Ceritinib was significantly reduced in the

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presence of Exo-Apo in comparison with that in the presence of Exo-Ctrl. Consistent with the data in Fig. 3 showing that Exo-Apo led to resistance to ALK inhibitor-induced cell death, these results suggest that Exo-Apo may transfer resistance to ALK-inhibition-induced apoptosis. Exo-Apo promotes growth of H3122 xenograft tumors To test whether the effects of exosomal ALK on H3122 cells in vitro could be replicated in vivo, tumor growth was assessed in a mouse xenograft model. Mice receiving Exo-Apo significantly increased tumor growth compared to those receiving Exo-Ctrl. Specifically, the major difference for xenograft tumor growth occurred from day 20 (Fig. 5A). Ceritinib treatment significantly inhibited tumor growth in mice receiving either Exo- Apo or Exo-Ctrl, but the extent of inhibition by Ceritinib was significantly less in Exo-Apo-treated mice than in Exo-Ctrltreated ones (Fig. 5A). To understand whether the signaling pathways probably mediating in vitro and in vivo effects of Exo-Apo are similar, we analyzed the levels of p-AKT, p-STAT3, and p-ERK in tumor xenografts from Exo-Apo-treated and Exo-Ctrl-treated mice. The tumor xenografts from Exo-Apo-treated mice exhibited upregulation of p-ALK, p-AKT, p-STAT3, and p-ERK in comparison with those from Exo-Ctrl-treated mice (Fig. 5B, C). It is possible that the resistance of recipient cells co-cultured with Exo-Apo to ALK inhibition may be due to overexpression of exosomal p-ALK, leading to over-activation of its downstream signaling pathways.

Discussion The dynamic interplay between the tumor and its microenvironment is a critical event that contributes to tumor progression and drug resistance. Microenvironment signals are provided by exosomes which play a key role in tumor host crosstalk (7, 8). Specifically, the functional

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capacity and compositions of exosomes are altered when the exosomes are exposed to cellular stressors or as tumors progress (9). We hereby reported that exosomes secreted from H3122 cells (Exo-Apo) harboring ALK after irradiation- induced apoptosis showed overexpression of p-ALK in comparison with the exosomes secreted from normal H3122 cells (Exo-Ctrl). Exo-Apo carried enriched p-ALK, which activated ALK pathways, and downstream ALT, STAT3, and ERK signaling pathways in recipient cells. These effects were accompanied by enhanced survival of recipient cells in vitro and growth of tumor xenograft in nude mice. Furthermore, transferring of p-ALK enriched exosomes into normal H3122 cells reduced sensitivity of these cells to ALK inhibitors, which may be related to increased ALK downstream signaling pathways. As a consequence, communication between apoptotic and non-apoptotic tumor cells via exosomes may transfer drug resistance among tumor cells and promote their survival. Irradiation-induced damage to DNA and other structures of a targeted cell is one of wellrecognized stress conditions influencing behavior of affected cells (16). Previous studies from different cancer cell models revealed that exosomes from irradiated cells significantly increase levels of proteins involving in transcription, translation, cell division, and cell signaling (17). Lehmann et al. showed that irradiated prostate cancer cells produced exosomes in vitro with elevated levels of B7-H3 (CD276), a diagnostic marker of prostate cancer (18). Here, we found that irradiation caused severe apoptosis in H3122 cells, evidenced by the upregulation of Caspase 3 and Bax, but reduction of Bcl-2 (Fig. 1). Accompanied with these changes, irradiation-treated H3122 cells also showed increased activation of ALK pathway in exosomes (Exo-Apo) released by these cells, evidenced by enhancement of phosphorylation of ALK. The finding extends the previous studies that cell stressors, such as, irradiation, could modify ALK pathway.

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ALK is a hotspot for the progress of tumor. Various rearrangements of ALK gene lead to the production of ALK fusion proteins that dimerize to constitutively activate ALK. Such ALK fusion proteins function as oncogenic drivers in the progress of both haematopoietic malignancies and solid tumors (2). Our data provide evidence to support the notion that mobilized ALK pathway carried by exosomes may drive progress of NSCLC. For instance, ExoApo from H3122 cells enhanced activation of ALK pathway in recipient H3122 cells (Fig. 2) and reduced apoptotic ratio in recipient H3122 cells with a marginal significance compared with Exo-Ctrl (Fig. 4). Xenograft of H3122 cells in nude mice exhibited faster growth after Exo-Apo treatment (Fig. 5). Although the roles of exosomes in lung cancer progression has been known (6, 7), our data suggest that ALK might be an important composite in exosomes to play these roles. We also observed that ALK inhibitors dose-dependently led to the death of ALK-positive H3122 cells (Fig. 3), support that patients with ALK-positive tumors may benefit from ALK inhibitors (19, 20). An exosome-associated increase in drug resistance has been demonstrated in several cell lines and drugs (21-23). For instance, ALK-rearranged tumors exhibit marked sensitivity to ALK inhibitors, such as, crizotinib, ceritinib, and TAE684 (24, 25). However, despite initial responses, acquired resistance to these targeted therapies inevitably leads to the progress of diseases. Our data revealed that addition of Exo-Apo in culture medium caused rightward-shift in the doseresponse curve of three ALK inhibitors in killing H3122 cells, suggesting that the resistance of tumor cells to ALK inhibitor could be carried by exosomes. The resistance could be related to the enhancement of ALK activity in Exo-Apo because Exo-Apo did not alter the maximal effects of ALK inhibitors. Clinical investigations show that approximately one-fourth of patients with ALK-rearranged NSCLC relapse (26). Multiple mechanisms have been proposed to interpret

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resistance of NSCLC with rearranged ALK to targeted therapy (27-29). It appears that, in addition to affect the phosphorylation of ALK, an increase in rearranged ALK gene copy numbers after cancer treatment can result in resistance. Patients that develop resistance to ALK inhibitor therapy have been identified to have 4-5 fold increase of rearranged ALK in NSCLC (26, 30). In line with this, it is possible that the resistance of recipient cells transferred with ExoApo to ALK inhibitor is due to overexpression of exosomal p-ALK, leading to over-activation of ALK downstream signaling in recipient cells. However, we cannot rule out the possibility that irradiation may induce new mutations in ALK domain or overactivation of the AKT, STAT-3 and ERK signaling cause bypass signaling (29, 31). Further investigations are warranted. In fact, in examining the effects of Exo-Apo on the sensitivity of recipience H3122 cells to ALK inhibitors, if we added too much ALK, which will bind to a considerable proportion of ALK inhibitors, it will apparently cause a right-ward shift of the dose-response curve. In our experiments, we used culture medium to dilute Exo-Apo and Exo-Ctrl to a final concentration of 100 µg protein / ml, then used this medium to culture recipient cells. We did not collect information required to calculate the number of cells that can provide this volume of exosomes. But it is certain that we added similar amount of Exo-Apo or Exo-Ctrl, quantified with total protein content, in the recipient cultures. As irradiation elevated ALK levels in Exo-Apo by only ~50% relative to Exo-Ctrl (Figure 1E), while Exo-Apo increased IC50 of ALK inhibitors by more than 2-fold relative to Exo-Ctrl (Figure 3), it is arguable that the shift of the dose-response relationship is merely due to the additional amount of ALK provided by Exo-Apo relative to Exo-Ctrl. Instead, ALK pathway-dependent events induced by Exo-Apo may play significant roles in reducing the sensitivity of H3122 cells to ALK inhibitors.

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Consistent with the notion that exosomes are key players for intercellular communicators, we found that Exo-Apo carrying mobilized ALK pathway enhanced the activity of ALK pathway in recipient cells. Evidence includes the enhancement of phosphorylation of AKT, STAT3, and ERK, three downstream effectors of ALK (2). Furthermore, addition of exosomes from ALKnegative PC9 cells treated with irradiation did not alter the phosphorylation status of AKT, STAT3, and ERK pathways in recipient H3122 cells (Fig. 2). Therefore, the enhancement of AKT, STAT3, and ERK pathways in recipient cells by Exo-Apo relies on ALK. Treatment of Exo-Apo also enhanced the activation of ALK pathway in xenograft of H3122 cells, providing a possibility that ALK and its downstream pathways could involve in the facilitation of tumor growth.

Conclusions Taken together, these results emphasized the importance of exosomes in cell-to-cell communication during the progress of NSCLC, suggesting that exosomes with ALK could be one of the mechanisms responsible for promoting tumor cell growth and reducing sensitivity to ALK inhibition.

Conflict of interest The authors declare no competing financial interest.

Acknowledgements

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This study was funded by the the Science and Technology Development Fund Project of Shenzhen (No. JCYJ 20150403091443310)

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Figure legends FIGURE 1. Irradiation induced apoptosis in H3122 cells and promoted p-ALK accumulation in exosomes. (A) Representative Western blot images of cell lysates prepared from H3122 cells exposed to 0, 3 and 6 Gy irradiation. (B) Statistical results of panel (A). Data are shown as means ± SEM. (C) Representative Western blot images of exosome markers, Alix and CD63, and Golgi marker, GM130. (D) Representative Western blot images and (E) statistical results of p-ALK and ALK in exosomes prepared from H3122 cells exposed to 0 and 6 Gy irradiation. (F)

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Representative Western blots and (G) statistical results of p-ALK and ALK in cell lysates prepared from H3122 cells exposed to 0 and 6 Gy radiation. Data were shown as means ± SEM in (B), (E), and (G). n = 6 in each condition. Statistical significance was assessed by one-way ANOVA with post-hoc Bonferroni test. ‘**’ and ‘***’ respectively represent p