IJC International Journal of Cancer

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1 IJC International Journal of Cancer Short Report Exosomes derived from mesenchymal non-small cell lung cancer cells promote chemoresistance Richard J. Lobb 1,2, Rosa van Amerongen 1, Adrian Wiegmans 1, Sunyoung Ham 1, Jill E. Larsen 2,3 and Andreas M oller 1,2 1 Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD, 4006, Australia 2 School of Medicine, University of Queensland, Brisbane, QLD, 4072, Australia 3 Oncogenomics Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD, 4006, Australia Non-small cell lung cancer (NSCLC) is the most common lung cancer type and the most common cause of mortality in lung cancer patients. NSCLC is often associated with resistance to chemotherapeutics and together with rapid metastatic spread, results in limited treatment options and poor patient survival. NSCLCs are heterogeneous, and consist of epithelial and mesenchymal NSCLC cells. Mesenchymal NSCLC cells are thought to be responsible for the chemoresistance phenotype, but if and how this phenotype can be transferred to other NSCLC cells is currently not known. We hypothesised that small extracellular vesicles, exosomes, secreted by mesenchymal NSCLC cells could potentially transfer the chemoresistance phenotype to surrounding epithelial NSCLC cells. To explore this possibility, we used a unique human bronchial epithelial cell (HBEC) model in which the parental cells were transformed from an epithelial to mesenchymal phenotype by introducing oncogenic alterations common in NSCLC. We found that exosomes derived from the oncogenically transformed, mesenchymal HBECs could transfer chemoresistance to the parental, epithelial HBECs and increase ZEB1 mrna, a master EMT transcription factor, in the recipient cells. Additionally, we demonstrate that exosomes from mesenchymal, but not epithelial HBECs contain the ZEB1 mrna, thereby providing a potential mechanism for the induction of a mesenchymal phenotype in recipient cells. Together, this work demonstrates for the first time that exosomes derived from mesenchymal, oncogenically transformed lung cells can transfer chemoresistance and mesenchymal phenotypes to recipient cells, likely via the transfer of ZEB1 mrna in exosomes. Lung cancer is the leading cause of cancer mortality worldwide, 1 largely due to metastasis and development of treatment resistance. Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer and most patients present with late stage disease and poor survival prospects. 1 The developmental epithelial-to-mesenchymal transition (EMT) process is the phenotypic depolarisation of epithelial cells to elongated mesenchymal cells, due to downregulation of epithelial properties and remodelling of the cytoskeleton to enhance migratory potential. 2 EMT has been associated with metastasis and acquired resistance to cancer therapies 3,4 in various cancers, including NSCLC. 5 EMT is the phenotypic depolarisation of epithelial cells to elongated mesenchymal Key words: non-small cell lung cancer, epithelial-to-mesenchymal transition, exosomes Grant sponsor: National Health and Medical Research Council; Grant numbers: NHMRC, APP DOI: /ijc History: Received 14 Nov 2016; Accepted 13 Mar 2017; Online 26 Apr 2017 Correspondence to: Andreas M oller; 300 Herston Road, Herston, QLD, Australia, 4006, Tel.: , Fax: , andreas.moller@qimrberghofer.edu.au cells, due to downregulation of epithelial properties and remodelling of the cytoskeleton to enhance migratory potential. 2 The loss of epithelial characteristics is associated with E-cadherin loss and gain of mesenchymal markers, such as vimentin. 2 E-cadherin is primarily regulated by canonical EMT transcription factors (Snail, Slug, Twist and Zeb1/2), that repress E-cadherin through interactions with the proximal region of the E-cadherin promoter. 2 Recently, it has been demonstrated that EMT is expendable for metastasis but is an essential step in the development of chemoresistance in breast and pancreatic cancer. 3,4 EMT reduces cancer cell proliferation, upregulates chemoresistance genes and is associated with suppression of drug transporters into the nucleus. 4 These changes in gene expression provide mesenchymal cancer cells with enhanced resistance to currently used chemotherapies. The role of EMT in NSCLC chemoresistance is less well defined. Several studies revealed that NSCLC cell lines that are more epithelial, with corresponding high E-cadherin expression protein levels, have been shown to be more sensitive to chemotherapeutics, while high Snail1 and Twist expression is involved in providing resistance to cisplatin. 6,7 This is of particular importance, as we have recently demonstrated that EMT transcription factors can be detected in NSCLC patients cancers at an early stage of disease. 5 Int. J. Cancer: 141, (2017) VC 2017 UICC

2 Lobb et al. 615 What s new? In non-small cell lung cancer (NSCLC), chemoresistant phenotypes are enhanced among mesenchymal cells, while some other NSCLC cell subpopulations are inherently sensitive to chemotherapy. Whether the latter cells can receive a resistance phenotype via transfer from mesenchymal cells remains unclear. To identify a possible transfer mechanism, the authors of this study investigated small extracellular vesicles known as exosomes. In a human bronchial epithelial cell model, they show that mesenchymal NSCLC-derived exosomes from chemoresistant cells are capable of transferring resistance to chemosensitive epithelial cells. Recipient cells exhibited increased mrna levels of the mesenchymal transcription factor ZEB1, suggesting a mechanism for chemoresistance induction. Given the heterogeneous nature of NSCLC, we aimed to determine if mesenchymal NSCLC cells could transfer a chemoresistant phenotype to NSCLC cells that are inherently sensitive to chemotherapies. Recently, exosomes have been implicated as key mediators of intercellular communication within the primary tumour microenvironment. 8,9 Exosomes are small membrane vesicles with a particle size of nm. 10 These small extracellular vesicles of endocytic origin are released into the extracellular milieu and can modify the phenotype of recipient cells. 11 Exosomes therefore serve as potent mediators of intercellular communication and have critical roles in tumorigenesis. We demonstrate that exosomes from mesenchymal NSCLC cells have an important function in the primary tumour through altering the chemotherapeutic sensitivity phenotype of recipient epithelial NSCLC. Our work provides novel and important insights into how primary tumour heterogeneity can promote tumorigenesis and chemotherapeutic drug resistance. Methods Cell culture Parental human bronchial epithelial cells (HBECs; 30KT) and HBECs with p53 knockdown, KRAS V12 overexpression and LKB1 knockdown (30KT p53/kras/lkb1 ) 12 were maintained in Keratinocyte serum-free media and incubated at 378C in 5% CO 2. Exosome isolation Exosomes were isolated as previously described. 13 Briefly, conditioned media was collected after 72 hrs and centrifuged at 300g at 48C for 10 min to remove floating cells. Supernatant was filtered (0.22 mm, Merck Millipore) to remove contaminating microvesicles and cell debris before centrifugation at 100,000g avg, 48C, 90 min. Exosome pellets were resuspended, pooled and centrifuged at 100,000g avg,48c, 90 min and resuspended in PBS. Tunable resistive pulse sensing (TRPS) The concentration and size distribution of particles was analysed with TRPS (qnano, Izon Science Ltd) using a NP100 nanopore at a 45 mm stretch. 70 nm carboxylated polystyrene beads ( particles/ml) were used to standardise concentration and size. Electron microscopy Exosomes were visualized using transmission electron microscopy (TEM) as described. 13 Briefly, exosome suspensions were fixed in paraformaldehyde and transferred onto Formvarcarbon coated electron microscopy grids and contrasted with uranyl-oxalate solution, ph 7, before transfer to methylcellulose-ua. Grids were observed with JEM 1,011 transmission electron microscope at 80 kv. Antibodies and reagents Antibodies used are CD63 (Abcam, ab8219), HSP70 (Transduction Laboratories, ), Calnexin (Cell Signaling Technology, 2679 S), Horseradish peroxidase (HRP)-conjugated secondary antibodies (Thermo Scientific). The methodology and primers used for the qrt-pcr have been described previously. 14 Chemotherapy dose curves Growth inhibition was measured as described. 15 In short, cells were analysed by MTS assay (Cell titer 96 AQueous Promega) and measured at 500 nm. Data was analyzed using GraphPad Prism and plotted as nonlinear regression line of best fit for IC 50. The IC 50 of gemcitabine was used in combination with cisplatin. Western blot analysis Western blots were performed as previously described. 16 Briefly, exosomes were lysed and proteins resolved by SDS- PAGE, transferred to polyvinylidene fluoride membranes, blocked in 5% non-fat powdered milk in PBS-T (0.5% Tween- 20) and probed with antibodies. Proteins were detected using X-ray film and enhanced chemiluminescence reagent (Amersham ECL Select). Statistical methods Statistical analyses were performed using Student s t test. All experiments were performed as a minimum of three independent replicates; p-values <0.05 were considered significant (*p < 0.05, **p < 0.001, ***p < ). Results The introduction of three common NSCLC oncogenic mutations (p53 knockdown, KRAS V12 over expression, LKB1 knockdown,

3 616 Exosomes derived from mesenchymal NSCLC cells promote chemoresistance 30KT p53/kras/lkb1 ) into an epithelial, typically rounded HBEC line (30KT) results in the cells exhibiting an elongated mesenchymal-like phenotype (Fig. 1a). These morphological changes are accompanied by decreased gene expression of CDH1 (the gene coding for E-cadherin), and increased expression of SNAI1, SNAI2, TWIST, ZEB1 and ZEB2 transcription factors (Fig. 1b). We have previously shown that EMT in another oncogenically transformed HBEC line (3KT) can induce a cancer stem cell-like phenotype, characterised by a CD24 /l 8 w CD44 1/ high population of cells. 5 This was also observed in 30KT, where the mesenchymal phenotype 30KT p53/kras/lkb1 is associated with a cancer stem cell-like phenotype (Figs. 1c and 1d). The assessment of the sensitivity of the parental 30KT and 30KT p53/kras/lkb1 cells to chemotherapies used in NSCLC showed that the mesenchymal 30KT p53/kras/lkb1 had a significantly higher resistance to cisplatin, gemcitabine, and the combination of cisplatin and gemcitabine (Fig. 1e). Together, these data show that in contrast to epithelial cells, mesenchymal cells are more stem cell-like and more resistant to common therapies used in NSCLC. Next, we isolated exosomes from both the 30KT and the 30KT p53/kras/lkb1 lines. Particles isolated from the cell supernatants have the exosome-typical cup-shaped morphology and lipid bilayer (Fig. 2a). TRPS analysis established that exosomes from both 30KT and 30KT p53/kras/lkb1 have a similar in size-distribution within the typical range for exosomes ( nm in diameter; Fig. 2b). Furthermore, the number of exosomes secreted by both cell lines was similar (Fig. 2c). Exosome preparation from 30KT and 30KT p53/kras/ LKB1 are positive for canonical exosome marker proteins HSP70 and CD63, and negative for the endoplasmic reticulum protein Calnexin (Fig. 2d). Taken together we show that exosomes from normal epithelial and malignant mesenchymal HBECs, have no morphological, size or concentration differences. To investigate if exosomes from the drug-resistant mesenchymal 30KT p53/kras/lkb1 cell line could promote chemoresistance in epithelial 30KT HBECs, exosome uptake was quantified by flow cytometry. DiD-labeled (5 and 50 mg/ml) 30KT p53/kras/lkb1 -derived exosomes were added to 30KT cells. At both concentrations, 100% of recipient cells took up exosomes in a time dependent manner (Fig. 2e). For subsequent experiments, we used 50 mg/ml DiD-labeled 30KT p53/kras/lkb1 -derived exosomes due to the 100% uptake observed in recipient cells after 12 hrs. Fluorescent microscopy was used to further ensure exosomes were taken up by 30KT cells. 30KT p53/kras/lkb1 -derived exosomes were internalised and localised around the nucleus at 24 hrs (Fig. 1f). We next postulated that exosomes derived from mesenchymal 30KT p53/kras/lkb1 cells would promote a chemoresistant phenotype in recipient epithelial 30KT HBECs. Indeed, 30KT cells previously exposed to 30KT p53/kras/lkb1 -derived exosomes 24 hrs before treatment with chemotherapy were significantly more resistant to IC 50 concentrations of gemcitabine, and the combination therapy of cisplatin and gemcitabine after 48 hrs compared with cells treated with epithelial 30KT-derived exosomes (Fig. 2g). We next hypothesised that exosomes derived from 30KT p53/kras/ LKB1 cells may promote EMT, given the role of EMT in chemoresistance. To determine if the acquired resistance phenotype was related to an increase in mesenchymal transcription factors, we assessed the expression of ZEB1, ZEB2, SNAI1, SNAI2 and TWIST1 (Fig. 3a). Interestingly, 30KT cells exposed to 30KT p53/kras/lkb1 -derived exosomes had a significantly increased expression of the transcription factor ZEB1 and TWIST1 (Fig. 3a). We hypothesised that either ZEB1 or TWIST1 mrna may be present in 30KT p53/kras/lkb1 -derived exosomes and responsible for the observed increase in ZEB1 and TWIST1 mrna in recipient 30KT cells. Interestingly, 30KT p53/kras/lkb1 -derived exosomes, but not 30KT-derived exosomes, contain ZEB1 mrna (Fig. 3b), whereas TWIST1 was not detected. To assess the functional impact of the transfer of exosomal ZEB1 mrna to recipient 30KT cells, we assessed the CD24/CD44 stem cell-like phenotype. We have previously shown that ZEB1 increases cell surface expression of CD44. 5 When comparing 30KT cells exposed to 30KT-derived and 30KT p53/kras/lkb1 -derived exosomes, the uptake of 30KT p53/kras/lkb1 -derived exosomes shifted the recipient cell population towards a stem cell-like CD24 l 8 w /CD44 high phenotype (Figs. 3c and 3d). Given that 30KT p53/kras/lkb1 -derived exosomes promoted stemness, and increased ZEB1 levels in recipient epithelial cells, we next questioned if ZEB1 can promote increased resistance to chemotherapy. To answer this, we overexpressed ZEB1 in an independent HBEC line (3KT). Compared to 3KT pmscv (control vector) cells, ZEB1 expression in 3KT ZEB1 was significantly elevated (Fig. 3e). This increased ZEB1 expression was also associated with a significant increase in resistance to cisplatin, gemcitabine and combination of cisplatin and gemcitabine, demonstrating that increased ZEB1 in lung cells can indeed provide a chemoresistant phenotype. Together, these data show for the first time that the transfer of exosomes of an oncogenically transformed, mesenchymal lung cell line can promote chemoresistance and a stem cell-like phenotype in epithelial lung cells. Discussion Small ( nm) vesicles were originally thought of as a mechanism to remove redundant proteins from a cell. 17 It is now however, becoming clear that exosomes have an integral role in the progression of cancer. 11,18,19 The capability of exosomes to travel between cell populations in the extracellular environment allows exosomes to modify recipient cell phenotypes, and can serve as potential novel biomarkers in numerous disease settings, including cancer. This has been highlighted recently, with a number of papers demonstrating the role of exosomes in predicting disease, 20 organotropic metastasis 19 and pre-metastatic niche formation. 11,18

4 Lobb et al. 617 Figure 1. Oncogenic mutations cause EMT and promote drug resistance in normal lung epithelial cells (HBECs). (a) p53 knockdown, KRAS V12 over expression, LKB1 knockdown cause morphological changes of epithelial 30KT cells into elongated mesenchymal-like 30KT p53/kras/lkb1 cells, size-bar 20 mm. (b) qpcr of HBEC cells demonstrate 30KT p53/kras/lkb1 have lower CDH1 expression, and increased EMT transcription factor expression. (c, d) EMT in HBEC cells is accompanied with a significant increase in the stem cell-like CD24 l 8 w CD44 high population. (e) Mesenchymal 30KT p53/kras/lkb1 cells are more resistant to cisplatin, gemcitabine and cisplatin gemcitabine combination therapy. N SEM, *p < 0.05, ***p <

5 618 Exosomes derived from mesenchymal NSCLC cells promote chemoresistance Figure 2. Exosomes derived from 30KT p53/kras/lkb1 cells promote chemoresistance. (a) Transmission electron microscopy demonstrates the presence of exosome vesicles (size bar nm). (b) TRPS analysis of exosomes isolations confirming the expected size range of nm in diameter. (c) No difference was observed in the total number of exosomes particles secreted from 30KT and 30KT p53/kras/lkb1 cells. (d) Western blot confirmation of exosomes markers indicates the presence of HSP70 and CD63 in exosome lysates (EL), but only Calnexin is present in cell lysates (CL) indicating pure preparations. (e) Time course internalisation of exosome uptake in 30KT cells of DiD-labelled exosomes. (f) Confocal microscopy demonstrating DiD-labelled exosome uptake in 30KT cells after 24 hrs. (g) After 48 hrs at previously established IC 50 values for 30KT cells, 30KT cells exposed to 30KT p53/kras/lkb1 exosomes are more resistant to gemcitabine and cisplatin gemcitabine combination therapy. N SEM, **p < 0.001, ***p <

6 Lobb et al. 619 Figure 3. 30KT p53/kras/lkb1 exosomes promote stemness in recipient epithelial cells. (a) qpcr of EMT transcription factors demonstrates that 30KT cells exposed to 30KT p53/kras/lkb1 exosomes have significantly higher ZEB1 and TWIST1 mrna compared to control cells that were exposed to 30KT exosomes. (b) qpcr of exosomal RNA demonstrates that 30KT p53/kras/lkb1 exosomes have ZEB1 mrna, whereas 30KT-derived exosomes are negative for ZEB1 mrna, moreover, TWIST1 was not detected in 30KT p53/kras/lkb1 or 30KT-derived exosomes (nd 5 not detected). (c,d) 30KT cells exposed to 30KT p53/kras/lkb1 exosomes have an increase in the population of CD24 l 8 w CD44 high cells, indicating a stem cell-like phenotype. (e) qpcr demonstrating that ZEB1 was overexpressed in HBEC 3KT cells (3KT-ZEB1) compared 3KT-pMSCV (control vector). (f) ZEB1 overexpression provided increased resistance to cisplatin, gemcitabine, and cisplatin gemcitabine combination. N SEM, *p < 0.05, **p < 0.001, ***p < [Color figure can be viewed at wileyonlinelibrary.com] Exosomes have been demonstrated to promote tumorigenesis and chemoresistance in a variety of cancers. Previously, oncogenesis promoting proteins have been demonstrated to be transferred between cancer cells through exosomes. Glioma cells can promote tumorigenesis through transfer of mutant epidermal growth factor receptor (EGFRvIII), which increases the expression of anti-apoptotic genes and enhances proliferation in recipient cells. 9 In support of these interesting findings, colon cancers cells transfer mutant KRAS via exosomes and promote three-dimensional growth of wild-type KRAS colon cancer cells. 8 As well as proteins, exosomes have been previously demonstrated to shuttle nucleic acids from a donor to recipient cells. 21 These findings suggest that exosomes have integral roles in the exchange of genetic information within the tumour microenvironment. Importantly, mrna transcripts transferred to recipient cells were shown to be active as they are translated into protein, establishing the functional nature of exosomes-derived RNA species. Our study further expands on the transfer of oncogenically promoting mrna into recipient cells. Here we show that mesenchymal-derived exosomes can transfer chemoresistant traits of donor cells to recipient cells. This results in a CSC-like phenotype and provides novel insights into how tumour heterogeneity may promote chemoresistance, and phenotypic changes of cancer cells in the primary tumour. This may be a result of the transfer of the EMT transcription factor ZEB1. To confirm ZEB1 is indeed involved in resistant mechanism in lung cells, we overexpressed ZEB1 in 3KT cells. We have previously demonstrated that overexpression of ZEB1 induces a CD24 l 8 w /CD44 high phenotype, 5 yet in this study we have revealed it also induces a chemoresistant phenotype (Fig. 1f). Overexpression of ZEB1 caused a more chemoresistant phenotype compared to exposure of exosomes, however, this is most likely due to the acute nature of exosome exposure, and the levels of ZEB1 expression in cells exposed to exosomes (Fig. 3a) compared to overexpression of ZEB1 (Fig. 3e). Cancer cells that have undergone EMT and display dedifferentiated, cancer stem cell-like phenotypes are inherently drug resistant, or are more capable of developing a chemoresistant phenotype. 22 EMT is generally considered a late event in cancer progression, but we and other investigations have shown EMT can occur in early stages of cancer progression. 5,23 Although CD24 l 8 w /CD44 high cell surface markers have not been definitively established as a marker of lung CSCs, we have previously demonstrated that CD24 l 8 w /

7 620 Exosomes derived from mesenchymal NSCLC cells promote chemoresistance CD44 high HBECs demonstrated increased malignant transformation compared to CD24 high /CD44 l 8 w cell populations. 5 We expand on this in the present study, to demonstrate CD24 l 8 w / CD44 high cells are more resistant to chemotherapy. This research further reveals that that CSC-like lung cells can modify and promote dedifferentiation of epithelial cells via exosomal communication, thereby providing novel insights into cellular transformation within the primary tumour, and insights into tumour progression and therapy. Given that tumours are heterogeneous, further understanding of the plasticity of cancer cells and how chemoresistance is promoted is needed to improve patient survival. 22 While we and others have previously demonstrated that exosomes can systemically alter secondary sites to enhance metastasis, 11,18 the current study highlights, for the first time, how exosomes can alter the response of lung epithelial cells to chemotherapeutic drugs. Given the unique proteomic and RNA content, and established protocols for the isolation of exosomes from body fluids, 13 exosomes represent a novel tool for biomarker analysis and therapeutic intervention. This study highlights the utility of understanding exosomal mediated transfer of mrna in detailing the clinical response of patients undergoing therapy. In conclusion, we have established that mesenchymal-derived exosomes as critical mediators of lung cancer pathogenesis, showing the importance of understanding exosomal communication in tumorigenesis. Acknowledgment The authors would like to thank the members of the Tumour Microenvironment Laboratory for their input and critical reading of the manuscript. References 1. Govindan R, Page N, Morgensztern D, et al. Changing epidemiology of small-cell lung cancer in the United States over the last 30 years: analysis of the surveillance, epidemiologic, and end results database. J Clin Oncol 2006;24: Thiery JP, Acloque H, Huang RY, et al. Epithelial-mesenchymal transitions in development and disease. Cell 2009;139: Fischer KR, Durrans A, Lee S, et al. Epithelial-tomesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature 2015;527: Zheng X, Carstens JL, Kim J, et al. 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