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STAT3 pathway
Lung Cancer 75 (2012) 167–177
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Mesenchymal stem cells enhance lung cancer initiation through activation of IL-6/JAK2/STAT3 pathway Han-Shui Hsu a,b,∗ , Jiun-Han Lin a , Tien-Wei Hsu a , Kelly Su a , Cheng-Wien Wang c , Kuang-Yao Yang d , Shih-Hwa Chiou e,f , Shih-Chieh Hung e,f,g,∗∗ a
Institute of Emergency and Critical Care Medicine, National Yang-Ming University School of Medicine, Taiwan Division of Thoracic Surgery, Department of Surgery, Taipei Veterans General Hospital, Taiwan c Department of Orthopedics, Ton-Yen General Hospital, Hsinchu, Taiwan d Department of Chest Medicine, Taipei Veterans General Hospital, National Yang-Ming University School of Medicine, Taiwan e Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taiwan f Stem Cell Laboratory, Department of Medical Research and Education, Taipei Veterans General Hospital, Taiwan g Institute of Pharmacology, National Yang-Ming University School of Medicine, Taipei, Taiwan b
a r t i c l e
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Article history: Received 18 March 2011 Received in revised form 14 June 2011 Accepted 2 July 2011 Keywords: IL6 JAK2 STAT3 Lung cancer Mesenchymal stem cell Tumor initiation
a b s t r a c t Background: The role of mesenchymal stem cells (MSCs) and IL-6 in lung cancer has not been welladdressed. We aimed to determine if MSCs can enhance the ability of tumor initiation of lung cancer cells, and link MSCs with activation of the IL-6/JAK2/STAT3 signaling pathway. Materials and methods: Lung cancer cell lines A549 and CL1-5 were directly or indirectly cocultured with MSCs. Spheres were defined as cell colonies with >50% area showing 3-dimensional structure and blurred cell margins. Cells without and with MSCs were injected into NOD/SCID mice. The percentage of tumor formation was determined. The influence of the IL-6/JAK2/STAT3 signaling pathway in cancer cell sphere formation and tumor growth were investigated. Results: A very small number of lung cancer cells, when mixed with otherwise non-tumorigenic MSCs, obtained de novo tumorigenicity when injected subcutaneously and allowed to form a tumor xenograft. Secretion of IL-6 from MSCs increased activation of the JAK2/STAT3 pathway in cancer cells, and enhanced sphere formation and tumor initiation. A reduced capacity of tumor formation of A549 and CL1-5 lung cancer cells when IL-6 was inhibited in MSCs or STAT3 was silenced in A549 and CL1-5 admixed with MSCs. Conclusions: Culture of A549 or CL1-5 lung cancer cells with MSCs increased sphere formation, drug resistance, and overexpression of pluripotency markers through activation of the IL-6/JAK2/STAT3 pathway. MSCs enhanced the capability of A549 and CL1-5 lung cancer cells to form tumors in immunodeficient mice. Blockade of the IL-6/JAK2/STAT3 pathway attenuated the capability of A549 and CL1-5 cells to form tumors. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Although tumorigenesis has been widely studied as a largely cell-autonomous process involving genetically transformed cancer cells, the role of stromal cells in the neoplastic microenvironment has been now intensively investigated. It has been observed that
∗ Corresponding author at: Institute of Emergency and Critical Care Medicine, National Yang-Ming University School of Medicine, No. 155, Sec. 2, Li-Nong Street, Taipei, Taiwan. Tel.: +886 2 28757546; fax: +886 2 28746193. ∗∗ Corresponding author at: Institute of Clinical Medicine, National Yang-Ming University School of Medicine, No. 155, Sec. 2, Li-Nong Street, Taipei, Taiwan. Tel.: +886 2 28757546; fax: +886 2 28746193. E-mail addresses: [email protected] (H.-S. Hsu), [email protected] (S.-C. Hung). 0169-5002/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2011.07.001
cancer development is associated with the occurrence of oncogenic events in tissue-resident adult stem cells and their microenvironment [1–3]. Tumors are comprised of a heterotypic array of malignant cells in communication with stromal cells. This tumor stroma is composed of endothelial cells, immune cells, and fibroblasts, which are thought to support the neoplastic properties of cancer cells [3]. Mesenchymal stem cells (MSCs) are a heterogenous population of non-hematopoietic precursor cells predominantly found in the bone marrow [4]. MSCs residing in tumor stroma have been reported to be associated with cancer metastasis. In 2007, Karnoub et al. [5] demonstrated that bone-marrow-derived human MSCs, when mixed with human breast carcinoma cells, can cause the cancer cells to greatly increase their metastatic potential. The enhanced metastatic ability is reversible, and is dependent on CCL5 signaling through the chemokine receptor CCR5 [5]. Recently,
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Guest et al. [6] reported that transgenic bone-marrow-derived stromal cells may indirectly contribute to the development of tumors derived from recipient mice by creating a transforming microenvironment. It has also been reported that MSCs can secret molecules to communicate and respond to other cell types, including several cytokines such as interleukin (IL)-1, IL-6, and IL-8 [7–9]. IL-6 is a multifunctional cytokine that was originally determined to be a regulator of immune and inflammatory responses [10]. IL-6 has been also reported to be associated with certain epithelial tumors [11–13]. Bachelot et al. [14] reported that serum IL-6 levels correlate with poor survival in patients with hormonerefractory metastatic breast cancer. Recently, Knupfer and Preiss [15] reviewed published results regarding colorectal cancer and concluded that circulating IL-6 might be a prognostic indicator in colorectal cancer. Yeh et al. [16] reported that higher levels of IL-6 were found in the malignant pleural effusions of patients with lung adenocarcinoma. IL-6 binds to a heterodimeric receptor containing the ligand-binding IL-6␣ chain and the common cytoline receptor signal-transducing subunit gp130 [17]. IL-6 receptor engagement leads to activation of the Janus kinases (JAKs) family of tyrosine kinases, which then stimulate pathways involving phosphatidylinositol 3-kinases (PI3Ks) and signal transducers and activators of transcription (STATs). STATs are a group of latent cytoplasmic transcription factors [17,18]. Activation of STAT3 plays an active role in the oncogenesis of a variety of tumors including leukemia, ovarian carcinoma, colon cancer, and lung adenocarcinoma [19–21]. The role of MSCs and IL-6 in lung cancer has not been well addressed. In this study, our aim was to determine if MSCs can enhance the tumor initiation ability of lung cancer cells, and link MSCs with activation of the IL-6/JAK2/STAT3 signaling pathway in growth of lung cancer. 2. Methods 2.1. Cell lines and culture conditions Lung cancer cell lines A549 and CL1-5 (gifts kindly provided by Dr. Yi-Ching Wang) were used in the study. Cells were grown in DMEM (Gibco, Grand Island, NY) or Ham’s F12 (Gibco, Grand Island, NY) containing 100 units/ml penicillin, 100 g/ml streptomycin, 4 mmol/L glutamine, and 10% fetal bovine serum (FBS; Gibco), in 37 ◦ C humidified atmosphere with 5% CO2 . 2.2. Mesenchymal stem cells Primary mesenchymal stem cells (MSCs) from different normal human volunteers were obtained from the Tulane Center for Distribution of Adult Stem Cells and were prepared and grown as described previously [22]. These immortalized or primary MSCs have been characterized to meet the definition of MSCs: plasticadherence, expression of MSC surface proteins such as CD29, CD44, CD90, CD73, CD105, CD166 and possession of differentiation potential into osteoblast, adipocyte and chondrocyte [23,24]. The passage number of MSCs used in the study is around 3–4. 2.3. Xenograft transplantation Non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice were purchased from the National Taiwan University Animal Facility (Taipei, Taiwan) and breed in specific pathogen-free conditions. The mice were used for experiments at 6–8 weeks of age. Tumor cells admixed without and with MSCs were injected subcutaneously for evaluation of tumor formation. When tumor nodules became palpable, usually about 2–7 mm in diameter, the length and width of the tumors were measured and tumor volume was calculated (tumor volume = length × width2 /2).
Tumor volume more than 4 mm3 was considered to be positive for tumor growth. 2.4. Sphere culture conditions and sphere counting assay Lung cancer cells were cultured in a modified tumor sphere medium (TSM):DMEM/F12 medium consisting of a chemically defined serum-free medium with N2 supplement human recombinant epidermal growth factor (EGF) (20 ng/ml; PeproTech) and basic fibroblast growth factor (bFGF) (10 ng/ml; PeproTech), and plated at a density of 104 cells/well in 6-well plates. Spheres were defined as 3-dimensional cell colonies with blurred cell margins. For cells treated with IL-6 (R&D Systems, Minneapolis, MN, USA) or indirectly co-cultured with MSCs, spheres were defined as cell colonies with >50% area showing 3-dimensional structure and blurred cell margins. 2.5. Co-culture procedures For direct co-culture, aliquots of MSC were mixed with lung cancer cells and seeded in 6-well plates. For indirect coculture, aliquots of MSC were plated on Falcon cell culture inserts on polyethylene terephthalate track-etched membranes with 23.1 mm diameter, 0.4 m pore size (Becton Dickinson). Lung cancer cells were seeded in the lower 6-well plates. The inserts and plates were added with growth medium of 1 ml and 2 ml respectively. Growth medium was replaced with sphere medium next day. Sphere medium was changed every three days for up to 12 days. 2.6. Immunohistochemistry For immunohistochemical staining, paraffin-embedded tumor sections were deparaffinized, rehydrated, and antigen retrieved by placing sections in Declere working solution (Cell Marque, Austin, TX) in a microwave oven for 20 min. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide. Residual enzymatic activity was removed by washes in PBS, and non-specific staining was blocked with Ultra V Block for 5 min (Thermo Fisher Scientific, Fremont, CA). Then, the sections were reacted with first antibodies overnight at 4 ◦ C, washed extensively with PBS, and reacted with corresponding biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) for 15 min at room temperature. The sections were then treated with streptavidin-peroxidase (LSAB Kit; Dako, Carpinteria, CA) followed by diaminobenzidine staining. Counterstaining was performed with Mayer’s hematoxylin. 2.7. Cytokine array and ELISA Samples of condition medium were centrifuged at 1500 × g for 10 min to remove cell debris, and then concentrated 30× using Amicon® Ultra Centrifugal Filter Devices (Millipore, Billerica, MA, USA). Membranes from a human protein cytokine array kit (Proteome ProfilerTM Array; R&D Systems) were blocked with a blocking buffer, and then incubated with 1 ml of the concentrated condition medium at room temperature for 1 h. The membranes were then washed with wash buffer, and assayed by chemiluminescence. To verify the results, the same samples were assayed with enzyme-linked immunosorbent assay (ELISA) kits for IL-6 (Quantikine® ; R&D). 2.8. Western blot analysis Cell extracts were prepared with M-PER (Pierce, Rockford, IL, USA) plus a protease inhibitor cocktail (HaltTM ; Pierce) and protein concentrations were determined using the bicinchoninic
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acid (BCA) assay (Pierce). Aliquots of protein lysates were separated on SDS–10% polyacrylamide gels and transferred to PVDF membrane filters, which were blocked with 5% blotting grade milk (Bio-Rad, Hercules, CA) in TBST (20 mM Tris–HCl [pH 7.6], 137 mM NaCl, 1% Tween 20). Membranes were then probed with the indicated primary antibodies (-actin; Sigma, St. Louis, MO, USA, Nanog (H-155), Oct4 (H-134), Sox2 (H-65); Santa Cruz Biotechnology, Santa Cruz, CA, USA, Phospho-Stat3 (Tyr705), Phospho-Jak2, Stat3, Jak2; Cell Signaling Technology Inc., Beverly, MA, USA, and IL-6; R&D Systems), reacted with corresponding secondary antibodies, and detected using a chemiluminescence assay (Millipore). Membranes were exposed to X-ray film to visualize the bands (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
2.9. Immunofluorescence For immunofluorescence study, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Trion X-100 for 30 min, then blocked with blocking buffer (5% heat inactivated serum and 0.1% Trion X-100 soluble in PBS) for 30 min, and then incubated with rabbit antibodies against human Oct3/4 (H-134; Santa Cruz Biotechnology), Sox-2 (H-65; Santa Cruz Biotechnology, CA), and Nanog (H-155; Santa Cruz Biotechnology) at appropriate dilutions overnight at 4 ◦ C, washed with PBS, and reacted with a secondary antibody labelled with Alexa Flour 488 (Molecular Probes). Immunofluorescence was examined by fluorescence microscopy.
2.10. TUNEL assay Cell numbers were determined using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, USA). Tumor cells and enriched MSCs (5 × 103 cells/well) were grown in 96-well plates, and treated with H2 O2 , cisplatin, gemcitabine, and quercetin at different dosages for 48 h. After incubation, the media was replaced with 50 l of MTT reagent (2 mg/ml) and incubated in 5% CO2 at 37 ◦ C for 2 h. Following incubation, the media were aspirated and DMSO (50 l) was added to each well. The optical density of each well was measured using a microplate reader (560 nm).
2.11. Lentiviral vector production and cell infection The expression plasmids and the bacteria clones for STAT3 siRNA (TRCN0000020840, TRCN0000020843) were provided by the National Science Council in Taiwan. Lentiviral production was performed by transfection of 293T cells using Lipofectamine 2000 (LF2000; Invitrogen, Carlsbad, CA, USA). Supernatants were collected 48 h after transfection and then filtered. Subconfluent cells were infected with lentivirus in the presence of 8 g/ml polybrene (Sigma–Aldrich). At 24 h post-infection, the medium was removed and replaced with fresh growth medium containing puromycin (4 g/ml), and selected for infected cells for 48 h.
2.12. Statistical analysis All values were expressed as mean ± standard deviation (SD). Independent t test was performed for comparison of data of independent samples. A probability (P) value < 0.05 was considered significant.
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3. Results 3.1. Mesenchymal stem cells promote tumor formation Five hundred A549 tumor cells coinjected with mesenchymal stem cells (MSCs) at concentrations of 5 × 103 to 5 × 104 , but not at 5 × 102 form tumors in immunodeficient mice (Fig. 1A), while 103 A549 cells or MSCs alone did not form tumors. A549 cells were found to be able to form tumors at a concentration up to 104 . Moreover, 500 A549 cells coinjected with 5 × 104 WI38 human lung fibroblasts did not form tumors. Tumors formed by either A549 cells alone or A549 cells with MSCs had the same histomorphology and expressed the same markers of primary lung tumors such as CK7, CK20, and TTF-1 (Fig. 1B), indicating that tumors were indeed of lung cancer origin [25,26]. These data suggest MSCs, but not lung mesenchymal cells, promote the capability of A549 lung cancer cells to form tumors. 3.2. Mesenchymal stem cells promote sphere formation of A549 and CL1-5 lung cancer cells and increase the expression of pluripotency MSC markers and chemoresistance in A549 lung cancer cells In in vitro lung sphere formation studies, coculture of MSCs with A549 and CL1-5 increased sphere formation in a dose dependent manner (Fig. 2). Fig. 2A demonstrates the increased sphere formation when A549 was indirectly cultured with MSCs. Fig. 2B demonstrates the increased number of spheres when A549 and CL1-5 were cultured with MSCs. Only slightly increased CL1-5 lung cancer cell sphere formation was observed when cultured with WI38 cells up to a concentration of 9 × 104 . Fig. 2C shows that in indirect culture, MSCs can still promote sphere formation of A549 and CL1-5 cells. Our previous studies demonstrated that the expression of pluripotency markers and the ability to resist chemotoxicity were increased in lung cancer stem cells [27,28]. Since coculture with MSCs increased tumor sphere formation, we then sought to determine whether lung cancer cells indirectly cocultured with MSCs can enhance the expression of the pluripotency makers. Western blotting showed increasing expression of pluripotency markers including Nanog, Sox2, and Oct4A in A549 cells mixed with MSCs, compared to A549 cells alone (Fig. 3A). Immunofluorescence study revealed the expression of pluripotency markers in A549 cells when A549 cells were indirectly cultured with MSCs (Fig. 3B). To investigate if A549 cells cultured with MSCs have the property of chemoresistance, DNA fragmentation assayed by the TUNEL technique was performed and revealed that apoptosis induced by a combination of cisplatin and gemcitabine was significantly reduced in A549 cells mixed with MSCs compared to A549 cells alone (Fig. 3C). 3.3. IL6 promotes sphere formation of A549 and CL1-5 cells, increases the expression of pluripotency MSC markers and chemoresistance in A549 cells, through activation of the IL-6/JAK2/STAT3 pathway To investigate which component of the MSCs conditioned medium can promote sphere formation in lung cancer cells, IL6, macrophage migration inhibitory factor (MIF), and plasminogen activator inhibitor (PAI)-1, which were apparently abundant in the MSC conditioned medium (Fig. 4A), were added to A549 and CL1-5 cells. We found that sphere formation was increased by adding IL6, but not MIF or PAI-1, to A549 and CL1-5 cells in a dose dependent manner (Fig. 4B). When IL-6 antibody was added to A549 cells cultured with MSCs or A549 cells cultured with MSCs transfected with IL-6 specific siRNA, sphere formation was significantly decreased
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Fig. 1. Mesenchymal stem cells promote tumor formation. (A) The results of tumor formation in immunodeficient mice when A549 lung cancer cells mixed with MSCs were injected subcutaneously. Five hundred A549 tumor cells cultured with MSCs up to a concentration of 5 × 104 showed an increased capability to form tumors in immunodeficient mice. (B) Immunohistochemistry study of the tumor blocks showed that tumors formed by either A549 cells alone or A549 cells with MSCs had the same histomorphology, and expressed the same markers as primary lung tumors, indicating that tumors were indeed of lung cancer origin. These data suggest MSCs promote tumor formation of lung cancer cells.
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Fig. 2. Mesenchymal stem cells promote sphere formation of A549 and CL1-5 lung cancer cells. (A) Increased sphere formation was observed when A549 cells were indirectly cultured with MSCs. The arrow represents sphere formation. (B) Comparison of the increased number of spheres when A549 cells and CL1-5 cells were cultured alone or with MSCs. Slightly increased CL1-5 lung cancer cell sphere formation was also observed when cultured with WI38 cells. (C) In indirect culture, MSCs can still promote sphere formation of A549 and CL1-5 cells.
(Fig. 4C and D), suggesting that IL-6 in MSCs was the main cytokine that promotes sphere formation in A549 and CL1-5 cells. When A549 cells were cultured with 293T cells with an IL-6 stable clone, increased sphere formation was also observed (Fig. 4E). Western blotting analysis showed increasing expression of embryonic markers including Nanog, Sox2, and Oct4A in A549 cells added with IL6, compared to A549 cells alone (Fig. 5A). Immunofluorescence study also revealed the expression of pluripotency markers in A549 cells when IL6 was added (Fig. 5B). To investigate if IL6 can enhance the chemoresistance of A549 cells, DNA fragmentation assayed by the TUNEL technique was performed and revealed that apoptosis induced by a combination of cisplatin and gemcitabine was also significantly reduced in A549 cells added with IL6, compared to A549 cells alone (Fig. 5C). To investigate whether the effect of IL-6 to promote sphere formation in lung cancer cells was through activation of the IL6/JAK2/STAT3 pathway, western blotting analysis was performed and showed that with IL-6, phosphorylated JAK2 and STAT3 were
activated in A549 cells (Fig. 6A). Fig. 6B demonstrates that when ag490 was added, phosphorylated JAK2 and STAT3 were subsequently inhibited. Fig. 6C shows that sphere formation also decreased when ag490 was added to A549 and CL1-5 cells. To further examine the involvement of STAT3, knockdown experiments to silence the expression of STAT3 using siRNAs were performed. We found that with STAT3 siRNAs, STAT3 expression was inactivated, and the sphere formation decreased in A549 and CL1-5 cells cultured with MSCs (Fig. 6D). 3.4. Reduced capability of tumor formation of MSCs in A549 lung cancer cells when IL-6 was inhibited in MSCs, or STAT3 was silenced in A549 cells mixed with MSCs Fig. 7A demonstrates the results of immunohistochemistry studies of the tumor blocks retrieved from the animals. Increased protein expression of phosphorylated JAK2 and STAT3 was observed in tumors formed by A549 cells mixed with MSCs, com-
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Fig. 3. A549 cells cultured with MSCs exhibited expression of pluripotency MSC markers and chemoresistance. (A) Western blotting analysis showed increased expression of pluripotency MSC markers including Nanog, Sox2, and Oct4A in A549 cells mixed with MSCs, as compared to A549 cells alone. (B) Immunofluorescence study revealed increased expression of pluripotency MSC markers in A549 cells when A549 cells were indirectly cultured with MSCs. (C) DNA fragmentation assayed by the TUNEL technique revealed that apoptosis induced by a combination of cisplatin and gemcitabine treatment was significantly reduced in A549 cells mixed with MSCs, as compared to A549 cells alone.
pared to tumors formed by A549 cells alone. Figure shows the results of tumor formation in immunodeficient mice injected with A549 cells mixed with MSCs, with or without IL-6 and STAT3 siRNAs. Tumors were formed in mice injected with A549 cells mixed with MSCs. However, when IL-6 siRNA or STAT3 siRNA was added to the A549 cells mixed with MSCs, the capability of MSCs to form tumors was blocked, suggesting that the capability of MSCs to promote tumor formation is through activation of the IL-6/JAK2/STAT3 pathway. Fig. 7C shows the results of tumor formation in immunodeficient mice when CL1-5 lung cancer cells were injected in the experiment; the results were the same as when A549 cells were used, although CL1-5 cells were less capable of forming tumors compared to the A549 cells. 4. Discussion MSCs are pluripotent progenitor cells that contribute to the maintenance and regeneration of a variety of connective tissues including bone, adipose, cartilage, and muscle [4]. Some studies
have suggested that MSCs can be recruited in large numbers to the stroma of developing tumors [29,30]. In human organs, there is a location or niche enriched with normal tissue or cancer stem cells that resides within a subpopulation of stromal cells, which are closely associated, and control the activity, of normal tissue or cancer stem cells. In these niches, MSCs secret molecules to communicate with and respond to other cell types. In vitro, MSCs can secret several cytokines and growth factors [31]. We demonstrated for the first time that MSCs increase sphere formation and overexpression of embryonic stem markers in A549 and CL1-5 lung cancer cells through activation of the IL-6/JAK2/STAT3 pathway, and that the capability of A549 and CL1-5 cells to form tumors in immunodeficient mice is also enhanced. Research on the relationship between MSCs and tumors is in progress. Coffelt et al. [32] reported that a proinflammatory peptide, LL-37, facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as proantigenic factor-expressing tumor stromal cells. Spaeth et al. [33] suggested that MSCs play an integral role in the development of
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Fig. 4. IL-6 promotes sphere formation of A549 and CL1-5 cells. (A) The results of analysis of the component in MSC conditioned medium. (B) Sphere formation was increased by adding IL-6 to A549 and CL1-5 cells in a dose dependent manner. (C and D) When IL-6 antibody or siRNA was added to A549 cells cultured with MSCs, sphere formation was significantly decreased, suggesting that IL-6 in MSCs was the main cytokine that promotes sphere formation in A549 and CL1-5 cells. (E) When A549 cells were cultured with 293T cells with an IL-6 stable clone, increased sphere formation was observed.
a mature, complex, physiological tumor microenvironment. They also pointed out that tumor associated fibroblasts derived from MSCs in the tumor microenvironment biologically impact tumor progression through the production of growth factors, cytokines, chemokines, matrix-degrading enzymes, and immune-modulatory
mechanisms [33]. In this study, we showed that when MSCs were cultured with lung cancer cells, increased sphere formation and overexpression of pluripotency markers including Nanog, Oct4A, and Sox2 were seen in lung cancer cells. Resistance to traditional chemotherapy of lung cancer was also seen in A549 lung cancer
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Fig. 5. A549 cells added with IL6 showed increased expression of pluripotency MSC markers and chemoresistance. (A) Western blotting analysis showed increased expression of pluripotency MSC markers including Nanog, Sox2, and Oct4A in A549 cells added with IL6, as compared to A549 cells alone. (B) Immunofluorescence study revealed expression of pluripotency MSC markers in A549 cells when IL6 was added. (C) DNA fragmentation assayed by the TUNEL technique revealed that apoptosis induced by a combination of cisplatin and gemcitabine treatment was also significantly reduced in A549 cells mixed with IL6, as compared to A549 cells alone.
cells cultured with MSCs. WI38, human lung fibroblast cells, were used in the study for a control experiment to demonstrate the enhancement of sphere formation by MSCs is more obvious and specific in MSCs rather than other tissues derived fibroblasts. We found that compared to MSCs, WI38 can only slightly increase the sphere formation of A549 and CL1-5 lung cancer cells at a concentration of 9 × 104 , while MSCs can significantly increase the sphere formation of cancer cells at a concentration of less than 1 × 104 . The exact mechanism about why WI38 can only slightly increased the sphere formation of lung cancer cells is not clear. We further demonstrated that blockade of IL-6 secreted from MSCs and the downstream pathway proteins, including JAK2 and STAT3, decreased sphere formation in A549 and CL1-1 cells, as well as the capability of tumor formation, compared to when A549 cells were indirectly cultured with MSCs, indicating that IL-6 is an important cytokine secreted from MSCs, and can enhance the tumor
initiation of A549 and CL1-5 lung cancer cells in immunodeficient mice. IL-6 has been reported to be associated with some epithelial cancers including breast, colon, and lung [14–16]. Increased levels of IL-6 have been observed in a variety of different tumors, and predict an adverse outcome [12]. As to the mechanisms of IL-6 involvement in cancer, in 2007 Sansone et al. [13] reported that IL-6 induces malignant features in Notch3-expressing stem cells from human ductal breast carcinoma and the normal mammary gland. Gao et al. [17] showed that mutant epidermal growth factor receptors (EGFRs) could activate the gp130/JAK/STAT3 pathway by means of IL-6 upregulation in primary human adenocarcinoma. In 2009, Colomiere et al. [20] reported that activation of STAT3 in high-grade ovarian carcinomas may occur directly through activation of EGFR or IL-6R, or indirectly through induction of IL-6R signaling. The results of our study suggest that MSCs from a tumor
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Fig. 6. Sphere formation of A549 and CL1-5 cells decreased when the IL6/JAK2/STAT3 pathway was inactivated. (A) Western blotting analysis showed that IL-6 activated phosphorylated JAK2 and STAT3 in A549 cells. (B) Western blotting analysis demonstrated that ag490 inhibited phosphorylated JAK2 and STAT3. (C) Sphere formation was decreased when ag490 was added to A549 and CL1-5 cells. STAT3 siRNAs and STAT3 expression was inactivated and sphere formation decreased in A549 and CL1-5 cells cultured with MSCs.
microenvironment may secret IL-6 to enhance tumor initiation in lung cancer through activation of the IL-6/JAK2/STAT3 pathway. Whether MSCs are involved in tumor development by secreting cytokines, or that MSCs are recruited to the microenvironment to facilitate the tumor progression is undetermined. It has been reported that the stroma has an active, oncogenic role in tumorigenesis [34,35]. In 2005, Orimo et al. [36] reported that carcinoma-associated fibroblasts extracted from human breast carcinoma promote the growth of breast carcinoma cells. In 2008,
Mishra et al. [37] reported that MSCs become activated and resemble carcinoma-associated myofibroblasts on prolonged exposure to condition medium from breast cancer cells. Contrary to our findings, Rattigan et al. [38] recently demonstrated that under hypoxic conditions, breast cancer cells secret high levels of IL-6, which serve to activate and attract MSCs. Further study to elucidate the interplay between MSCs and the tumor microenvironment is important. In summary, our data showed that when A549 or CL1-5 lung cancer cells were cultured with MSCs, increased sphere forma-
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Fig. 7. Reduced capability of tumor formation of MSCs in A549 lung cancer cells when IL-6 was inhibited in MSCs or STAT3 was silenced in A549 cells mixed with MSCs. (A) Immunohistochemistry studies of tumor blocks retrieved from the animals showed that increased protein expression of phosphorylated JAK2 and STAT3 was observed in tumors formed by A549 cells mixed with MSCs, compared to tumors formed by A549 cells alone. (B) Tumors were formed in mice injected with A549 cells mixed with MSCs. However, when IL-6 siRNA or STAT3 siRNA was added to the A549 cells mixed with MSCs, the capacity of MSCs to form tumors was blocked, suggesting that the capacity of MSCs to promote tumor formation is through activation of the IL-6/JAK2/STAT3 pathway. (C) Tumor formation in immunodeficient mice when CL1-5 lung cancer cells were injected in the study, showing the same results as when A549 cells were used, although CL1-5 cells were less capable of forming tumors as compared to A549 cells.
tion, drug resistance and overexpression of pluripotency markers were observed, as compared to A549 or CL1-5 cells cultured alone, through activation of the IL-6/JAK2/STAT3 pathway. In in vivo study, MSCs can enhance the capability of A549 and CL1-5 lung cancer cells to form tumors in immunodeficient mice. Upon blockade of the IL-6/JAK2/STAT3 pathway, the capability of A549 and CL1-5 lung cancer cells to form tumors was attenuated. Further investigation to study the role of MSCs and the secretion of cytokines in lung cancer tumorigenesis is mandatory. Conflict of interest The authors declare no conflicts of interest.
Acknowledgements This work was assisted in part by the Division of Experimental Surgery of the Department of Surgery, Taipei Veterans General Hospital, and supported by the Grant V99C1-035 from Taipei Veterans General Hospital to Han-Shui Hsu and the Lung Cancer Foundation in Memory of Doctor KS Lu. References [1] Tlsty TD. Stromal cells can contribute oncogenic signals. Semin Cancer Biol 2001;11:97–104. [2] Elenbaas B, Weinberg RA. Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation. Exp Cell Res 2001;264:169–84.
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Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
Mesenchymal stem cells enhance lung cancer initiation through activation of IL-6/JAK2/STAT3 pathway Han-Shui Hsu a,b,∗ , Jiun-Han Lin a , Tien-Wei Hsu a , Kelly Su a , Cheng-Wien Wang c , Kuang-Yao Yang d , Shih-Hwa Chiou e,f , Shih-Chieh Hung e,f,g,∗∗ a
Institute of Emergency and Critical Care Medicine, National Yang-Ming University School of Medicine, Taiwan Division of Thoracic Surgery, Department of Surgery, Taipei Veterans General Hospital, Taiwan c Department of Orthopedics, Ton-Yen General Hospital, Hsinchu, Taiwan d Department of Chest Medicine, Taipei Veterans General Hospital, National Yang-Ming University School of Medicine, Taiwan e Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taiwan f Stem Cell Laboratory, Department of Medical Research and Education, Taipei Veterans General Hospital, Taiwan g Institute of Pharmacology, National Yang-Ming University School of Medicine, Taipei, Taiwan b
a r t i c l e
i n f o
Article history: Received 18 March 2011 Received in revised form 14 June 2011 Accepted 2 July 2011 Keywords: IL6 JAK2 STAT3 Lung cancer Mesenchymal stem cell Tumor initiation
a b s t r a c t Background: The role of mesenchymal stem cells (MSCs) and IL-6 in lung cancer has not been welladdressed. We aimed to determine if MSCs can enhance the ability of tumor initiation of lung cancer cells, and link MSCs with activation of the IL-6/JAK2/STAT3 signaling pathway. Materials and methods: Lung cancer cell lines A549 and CL1-5 were directly or indirectly cocultured with MSCs. Spheres were defined as cell colonies with >50% area showing 3-dimensional structure and blurred cell margins. Cells without and with MSCs were injected into NOD/SCID mice. The percentage of tumor formation was determined. The influence of the IL-6/JAK2/STAT3 signaling pathway in cancer cell sphere formation and tumor growth were investigated. Results: A very small number of lung cancer cells, when mixed with otherwise non-tumorigenic MSCs, obtained de novo tumorigenicity when injected subcutaneously and allowed to form a tumor xenograft. Secretion of IL-6 from MSCs increased activation of the JAK2/STAT3 pathway in cancer cells, and enhanced sphere formation and tumor initiation. A reduced capacity of tumor formation of A549 and CL1-5 lung cancer cells when IL-6 was inhibited in MSCs or STAT3 was silenced in A549 and CL1-5 admixed with MSCs. Conclusions: Culture of A549 or CL1-5 lung cancer cells with MSCs increased sphere formation, drug resistance, and overexpression of pluripotency markers through activation of the IL-6/JAK2/STAT3 pathway. MSCs enhanced the capability of A549 and CL1-5 lung cancer cells to form tumors in immunodeficient mice. Blockade of the IL-6/JAK2/STAT3 pathway attenuated the capability of A549 and CL1-5 cells to form tumors. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Although tumorigenesis has been widely studied as a largely cell-autonomous process involving genetically transformed cancer cells, the role of stromal cells in the neoplastic microenvironment has been now intensively investigated. It has been observed that
∗ Corresponding author at: Institute of Emergency and Critical Care Medicine, National Yang-Ming University School of Medicine, No. 155, Sec. 2, Li-Nong Street, Taipei, Taiwan. Tel.: +886 2 28757546; fax: +886 2 28746193. ∗∗ Corresponding author at: Institute of Clinical Medicine, National Yang-Ming University School of Medicine, No. 155, Sec. 2, Li-Nong Street, Taipei, Taiwan. Tel.: +886 2 28757546; fax: +886 2 28746193. E-mail addresses: [email protected] (H.-S. Hsu), [email protected] (S.-C. Hung). 0169-5002/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2011.07.001
cancer development is associated with the occurrence of oncogenic events in tissue-resident adult stem cells and their microenvironment [1–3]. Tumors are comprised of a heterotypic array of malignant cells in communication with stromal cells. This tumor stroma is composed of endothelial cells, immune cells, and fibroblasts, which are thought to support the neoplastic properties of cancer cells [3]. Mesenchymal stem cells (MSCs) are a heterogenous population of non-hematopoietic precursor cells predominantly found in the bone marrow [4]. MSCs residing in tumor stroma have been reported to be associated with cancer metastasis. In 2007, Karnoub et al. [5] demonstrated that bone-marrow-derived human MSCs, when mixed with human breast carcinoma cells, can cause the cancer cells to greatly increase their metastatic potential. The enhanced metastatic ability is reversible, and is dependent on CCL5 signaling through the chemokine receptor CCR5 [5]. Recently,
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Guest et al. [6] reported that transgenic bone-marrow-derived stromal cells may indirectly contribute to the development of tumors derived from recipient mice by creating a transforming microenvironment. It has also been reported that MSCs can secret molecules to communicate and respond to other cell types, including several cytokines such as interleukin (IL)-1, IL-6, and IL-8 [7–9]. IL-6 is a multifunctional cytokine that was originally determined to be a regulator of immune and inflammatory responses [10]. IL-6 has been also reported to be associated with certain epithelial tumors [11–13]. Bachelot et al. [14] reported that serum IL-6 levels correlate with poor survival in patients with hormonerefractory metastatic breast cancer. Recently, Knupfer and Preiss [15] reviewed published results regarding colorectal cancer and concluded that circulating IL-6 might be a prognostic indicator in colorectal cancer. Yeh et al. [16] reported that higher levels of IL-6 were found in the malignant pleural effusions of patients with lung adenocarcinoma. IL-6 binds to a heterodimeric receptor containing the ligand-binding IL-6␣ chain and the common cytoline receptor signal-transducing subunit gp130 [17]. IL-6 receptor engagement leads to activation of the Janus kinases (JAKs) family of tyrosine kinases, which then stimulate pathways involving phosphatidylinositol 3-kinases (PI3Ks) and signal transducers and activators of transcription (STATs). STATs are a group of latent cytoplasmic transcription factors [17,18]. Activation of STAT3 plays an active role in the oncogenesis of a variety of tumors including leukemia, ovarian carcinoma, colon cancer, and lung adenocarcinoma [19–21]. The role of MSCs and IL-6 in lung cancer has not been well addressed. In this study, our aim was to determine if MSCs can enhance the tumor initiation ability of lung cancer cells, and link MSCs with activation of the IL-6/JAK2/STAT3 signaling pathway in growth of lung cancer. 2. Methods 2.1. Cell lines and culture conditions Lung cancer cell lines A549 and CL1-5 (gifts kindly provided by Dr. Yi-Ching Wang) were used in the study. Cells were grown in DMEM (Gibco, Grand Island, NY) or Ham’s F12 (Gibco, Grand Island, NY) containing 100 units/ml penicillin, 100 g/ml streptomycin, 4 mmol/L glutamine, and 10% fetal bovine serum (FBS; Gibco), in 37 ◦ C humidified atmosphere with 5% CO2 . 2.2. Mesenchymal stem cells Primary mesenchymal stem cells (MSCs) from different normal human volunteers were obtained from the Tulane Center for Distribution of Adult Stem Cells and were prepared and grown as described previously [22]. These immortalized or primary MSCs have been characterized to meet the definition of MSCs: plasticadherence, expression of MSC surface proteins such as CD29, CD44, CD90, CD73, CD105, CD166 and possession of differentiation potential into osteoblast, adipocyte and chondrocyte [23,24]. The passage number of MSCs used in the study is around 3–4. 2.3. Xenograft transplantation Non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice were purchased from the National Taiwan University Animal Facility (Taipei, Taiwan) and breed in specific pathogen-free conditions. The mice were used for experiments at 6–8 weeks of age. Tumor cells admixed without and with MSCs were injected subcutaneously for evaluation of tumor formation. When tumor nodules became palpable, usually about 2–7 mm in diameter, the length and width of the tumors were measured and tumor volume was calculated (tumor volume = length × width2 /2).
Tumor volume more than 4 mm3 was considered to be positive for tumor growth. 2.4. Sphere culture conditions and sphere counting assay Lung cancer cells were cultured in a modified tumor sphere medium (TSM):DMEM/F12 medium consisting of a chemically defined serum-free medium with N2 supplement human recombinant epidermal growth factor (EGF) (20 ng/ml; PeproTech) and basic fibroblast growth factor (bFGF) (10 ng/ml; PeproTech), and plated at a density of 104 cells/well in 6-well plates. Spheres were defined as 3-dimensional cell colonies with blurred cell margins. For cells treated with IL-6 (R&D Systems, Minneapolis, MN, USA) or indirectly co-cultured with MSCs, spheres were defined as cell colonies with >50% area showing 3-dimensional structure and blurred cell margins. 2.5. Co-culture procedures For direct co-culture, aliquots of MSC were mixed with lung cancer cells and seeded in 6-well plates. For indirect coculture, aliquots of MSC were plated on Falcon cell culture inserts on polyethylene terephthalate track-etched membranes with 23.1 mm diameter, 0.4 m pore size (Becton Dickinson). Lung cancer cells were seeded in the lower 6-well plates. The inserts and plates were added with growth medium of 1 ml and 2 ml respectively. Growth medium was replaced with sphere medium next day. Sphere medium was changed every three days for up to 12 days. 2.6. Immunohistochemistry For immunohistochemical staining, paraffin-embedded tumor sections were deparaffinized, rehydrated, and antigen retrieved by placing sections in Declere working solution (Cell Marque, Austin, TX) in a microwave oven for 20 min. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide. Residual enzymatic activity was removed by washes in PBS, and non-specific staining was blocked with Ultra V Block for 5 min (Thermo Fisher Scientific, Fremont, CA). Then, the sections were reacted with first antibodies overnight at 4 ◦ C, washed extensively with PBS, and reacted with corresponding biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) for 15 min at room temperature. The sections were then treated with streptavidin-peroxidase (LSAB Kit; Dako, Carpinteria, CA) followed by diaminobenzidine staining. Counterstaining was performed with Mayer’s hematoxylin. 2.7. Cytokine array and ELISA Samples of condition medium were centrifuged at 1500 × g for 10 min to remove cell debris, and then concentrated 30× using Amicon® Ultra Centrifugal Filter Devices (Millipore, Billerica, MA, USA). Membranes from a human protein cytokine array kit (Proteome ProfilerTM Array; R&D Systems) were blocked with a blocking buffer, and then incubated with 1 ml of the concentrated condition medium at room temperature for 1 h. The membranes were then washed with wash buffer, and assayed by chemiluminescence. To verify the results, the same samples were assayed with enzyme-linked immunosorbent assay (ELISA) kits for IL-6 (Quantikine® ; R&D). 2.8. Western blot analysis Cell extracts were prepared with M-PER (Pierce, Rockford, IL, USA) plus a protease inhibitor cocktail (HaltTM ; Pierce) and protein concentrations were determined using the bicinchoninic
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acid (BCA) assay (Pierce). Aliquots of protein lysates were separated on SDS–10% polyacrylamide gels and transferred to PVDF membrane filters, which were blocked with 5% blotting grade milk (Bio-Rad, Hercules, CA) in TBST (20 mM Tris–HCl [pH 7.6], 137 mM NaCl, 1% Tween 20). Membranes were then probed with the indicated primary antibodies (-actin; Sigma, St. Louis, MO, USA, Nanog (H-155), Oct4 (H-134), Sox2 (H-65); Santa Cruz Biotechnology, Santa Cruz, CA, USA, Phospho-Stat3 (Tyr705), Phospho-Jak2, Stat3, Jak2; Cell Signaling Technology Inc., Beverly, MA, USA, and IL-6; R&D Systems), reacted with corresponding secondary antibodies, and detected using a chemiluminescence assay (Millipore). Membranes were exposed to X-ray film to visualize the bands (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
2.9. Immunofluorescence For immunofluorescence study, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Trion X-100 for 30 min, then blocked with blocking buffer (5% heat inactivated serum and 0.1% Trion X-100 soluble in PBS) for 30 min, and then incubated with rabbit antibodies against human Oct3/4 (H-134; Santa Cruz Biotechnology), Sox-2 (H-65; Santa Cruz Biotechnology, CA), and Nanog (H-155; Santa Cruz Biotechnology) at appropriate dilutions overnight at 4 ◦ C, washed with PBS, and reacted with a secondary antibody labelled with Alexa Flour 488 (Molecular Probes). Immunofluorescence was examined by fluorescence microscopy.
2.10. TUNEL assay Cell numbers were determined using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, USA). Tumor cells and enriched MSCs (5 × 103 cells/well) were grown in 96-well plates, and treated with H2 O2 , cisplatin, gemcitabine, and quercetin at different dosages for 48 h. After incubation, the media was replaced with 50 l of MTT reagent (2 mg/ml) and incubated in 5% CO2 at 37 ◦ C for 2 h. Following incubation, the media were aspirated and DMSO (50 l) was added to each well. The optical density of each well was measured using a microplate reader (560 nm).
2.11. Lentiviral vector production and cell infection The expression plasmids and the bacteria clones for STAT3 siRNA (TRCN0000020840, TRCN0000020843) were provided by the National Science Council in Taiwan. Lentiviral production was performed by transfection of 293T cells using Lipofectamine 2000 (LF2000; Invitrogen, Carlsbad, CA, USA). Supernatants were collected 48 h after transfection and then filtered. Subconfluent cells were infected with lentivirus in the presence of 8 g/ml polybrene (Sigma–Aldrich). At 24 h post-infection, the medium was removed and replaced with fresh growth medium containing puromycin (4 g/ml), and selected for infected cells for 48 h.
2.12. Statistical analysis All values were expressed as mean ± standard deviation (SD). Independent t test was performed for comparison of data of independent samples. A probability (P) value < 0.05 was considered significant.
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3. Results 3.1. Mesenchymal stem cells promote tumor formation Five hundred A549 tumor cells coinjected with mesenchymal stem cells (MSCs) at concentrations of 5 × 103 to 5 × 104 , but not at 5 × 102 form tumors in immunodeficient mice (Fig. 1A), while 103 A549 cells or MSCs alone did not form tumors. A549 cells were found to be able to form tumors at a concentration up to 104 . Moreover, 500 A549 cells coinjected with 5 × 104 WI38 human lung fibroblasts did not form tumors. Tumors formed by either A549 cells alone or A549 cells with MSCs had the same histomorphology and expressed the same markers of primary lung tumors such as CK7, CK20, and TTF-1 (Fig. 1B), indicating that tumors were indeed of lung cancer origin [25,26]. These data suggest MSCs, but not lung mesenchymal cells, promote the capability of A549 lung cancer cells to form tumors. 3.2. Mesenchymal stem cells promote sphere formation of A549 and CL1-5 lung cancer cells and increase the expression of pluripotency MSC markers and chemoresistance in A549 lung cancer cells In in vitro lung sphere formation studies, coculture of MSCs with A549 and CL1-5 increased sphere formation in a dose dependent manner (Fig. 2). Fig. 2A demonstrates the increased sphere formation when A549 was indirectly cultured with MSCs. Fig. 2B demonstrates the increased number of spheres when A549 and CL1-5 were cultured with MSCs. Only slightly increased CL1-5 lung cancer cell sphere formation was observed when cultured with WI38 cells up to a concentration of 9 × 104 . Fig. 2C shows that in indirect culture, MSCs can still promote sphere formation of A549 and CL1-5 cells. Our previous studies demonstrated that the expression of pluripotency markers and the ability to resist chemotoxicity were increased in lung cancer stem cells [27,28]. Since coculture with MSCs increased tumor sphere formation, we then sought to determine whether lung cancer cells indirectly cocultured with MSCs can enhance the expression of the pluripotency makers. Western blotting showed increasing expression of pluripotency markers including Nanog, Sox2, and Oct4A in A549 cells mixed with MSCs, compared to A549 cells alone (Fig. 3A). Immunofluorescence study revealed the expression of pluripotency markers in A549 cells when A549 cells were indirectly cultured with MSCs (Fig. 3B). To investigate if A549 cells cultured with MSCs have the property of chemoresistance, DNA fragmentation assayed by the TUNEL technique was performed and revealed that apoptosis induced by a combination of cisplatin and gemcitabine was significantly reduced in A549 cells mixed with MSCs compared to A549 cells alone (Fig. 3C). 3.3. IL6 promotes sphere formation of A549 and CL1-5 cells, increases the expression of pluripotency MSC markers and chemoresistance in A549 cells, through activation of the IL-6/JAK2/STAT3 pathway To investigate which component of the MSCs conditioned medium can promote sphere formation in lung cancer cells, IL6, macrophage migration inhibitory factor (MIF), and plasminogen activator inhibitor (PAI)-1, which were apparently abundant in the MSC conditioned medium (Fig. 4A), were added to A549 and CL1-5 cells. We found that sphere formation was increased by adding IL6, but not MIF or PAI-1, to A549 and CL1-5 cells in a dose dependent manner (Fig. 4B). When IL-6 antibody was added to A549 cells cultured with MSCs or A549 cells cultured with MSCs transfected with IL-6 specific siRNA, sphere formation was significantly decreased
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Fig. 1. Mesenchymal stem cells promote tumor formation. (A) The results of tumor formation in immunodeficient mice when A549 lung cancer cells mixed with MSCs were injected subcutaneously. Five hundred A549 tumor cells cultured with MSCs up to a concentration of 5 × 104 showed an increased capability to form tumors in immunodeficient mice. (B) Immunohistochemistry study of the tumor blocks showed that tumors formed by either A549 cells alone or A549 cells with MSCs had the same histomorphology, and expressed the same markers as primary lung tumors, indicating that tumors were indeed of lung cancer origin. These data suggest MSCs promote tumor formation of lung cancer cells.
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Fig. 2. Mesenchymal stem cells promote sphere formation of A549 and CL1-5 lung cancer cells. (A) Increased sphere formation was observed when A549 cells were indirectly cultured with MSCs. The arrow represents sphere formation. (B) Comparison of the increased number of spheres when A549 cells and CL1-5 cells were cultured alone or with MSCs. Slightly increased CL1-5 lung cancer cell sphere formation was also observed when cultured with WI38 cells. (C) In indirect culture, MSCs can still promote sphere formation of A549 and CL1-5 cells.
(Fig. 4C and D), suggesting that IL-6 in MSCs was the main cytokine that promotes sphere formation in A549 and CL1-5 cells. When A549 cells were cultured with 293T cells with an IL-6 stable clone, increased sphere formation was also observed (Fig. 4E). Western blotting analysis showed increasing expression of embryonic markers including Nanog, Sox2, and Oct4A in A549 cells added with IL6, compared to A549 cells alone (Fig. 5A). Immunofluorescence study also revealed the expression of pluripotency markers in A549 cells when IL6 was added (Fig. 5B). To investigate if IL6 can enhance the chemoresistance of A549 cells, DNA fragmentation assayed by the TUNEL technique was performed and revealed that apoptosis induced by a combination of cisplatin and gemcitabine was also significantly reduced in A549 cells added with IL6, compared to A549 cells alone (Fig. 5C). To investigate whether the effect of IL-6 to promote sphere formation in lung cancer cells was through activation of the IL6/JAK2/STAT3 pathway, western blotting analysis was performed and showed that with IL-6, phosphorylated JAK2 and STAT3 were
activated in A549 cells (Fig. 6A). Fig. 6B demonstrates that when ag490 was added, phosphorylated JAK2 and STAT3 were subsequently inhibited. Fig. 6C shows that sphere formation also decreased when ag490 was added to A549 and CL1-5 cells. To further examine the involvement of STAT3, knockdown experiments to silence the expression of STAT3 using siRNAs were performed. We found that with STAT3 siRNAs, STAT3 expression was inactivated, and the sphere formation decreased in A549 and CL1-5 cells cultured with MSCs (Fig. 6D). 3.4. Reduced capability of tumor formation of MSCs in A549 lung cancer cells when IL-6 was inhibited in MSCs, or STAT3 was silenced in A549 cells mixed with MSCs Fig. 7A demonstrates the results of immunohistochemistry studies of the tumor blocks retrieved from the animals. Increased protein expression of phosphorylated JAK2 and STAT3 was observed in tumors formed by A549 cells mixed with MSCs, com-
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Fig. 3. A549 cells cultured with MSCs exhibited expression of pluripotency MSC markers and chemoresistance. (A) Western blotting analysis showed increased expression of pluripotency MSC markers including Nanog, Sox2, and Oct4A in A549 cells mixed with MSCs, as compared to A549 cells alone. (B) Immunofluorescence study revealed increased expression of pluripotency MSC markers in A549 cells when A549 cells were indirectly cultured with MSCs. (C) DNA fragmentation assayed by the TUNEL technique revealed that apoptosis induced by a combination of cisplatin and gemcitabine treatment was significantly reduced in A549 cells mixed with MSCs, as compared to A549 cells alone.
pared to tumors formed by A549 cells alone. Figure shows the results of tumor formation in immunodeficient mice injected with A549 cells mixed with MSCs, with or without IL-6 and STAT3 siRNAs. Tumors were formed in mice injected with A549 cells mixed with MSCs. However, when IL-6 siRNA or STAT3 siRNA was added to the A549 cells mixed with MSCs, the capability of MSCs to form tumors was blocked, suggesting that the capability of MSCs to promote tumor formation is through activation of the IL-6/JAK2/STAT3 pathway. Fig. 7C shows the results of tumor formation in immunodeficient mice when CL1-5 lung cancer cells were injected in the experiment; the results were the same as when A549 cells were used, although CL1-5 cells were less capable of forming tumors compared to the A549 cells. 4. Discussion MSCs are pluripotent progenitor cells that contribute to the maintenance and regeneration of a variety of connective tissues including bone, adipose, cartilage, and muscle [4]. Some studies
have suggested that MSCs can be recruited in large numbers to the stroma of developing tumors [29,30]. In human organs, there is a location or niche enriched with normal tissue or cancer stem cells that resides within a subpopulation of stromal cells, which are closely associated, and control the activity, of normal tissue or cancer stem cells. In these niches, MSCs secret molecules to communicate with and respond to other cell types. In vitro, MSCs can secret several cytokines and growth factors [31]. We demonstrated for the first time that MSCs increase sphere formation and overexpression of embryonic stem markers in A549 and CL1-5 lung cancer cells through activation of the IL-6/JAK2/STAT3 pathway, and that the capability of A549 and CL1-5 cells to form tumors in immunodeficient mice is also enhanced. Research on the relationship between MSCs and tumors is in progress. Coffelt et al. [32] reported that a proinflammatory peptide, LL-37, facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as proantigenic factor-expressing tumor stromal cells. Spaeth et al. [33] suggested that MSCs play an integral role in the development of
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Fig. 4. IL-6 promotes sphere formation of A549 and CL1-5 cells. (A) The results of analysis of the component in MSC conditioned medium. (B) Sphere formation was increased by adding IL-6 to A549 and CL1-5 cells in a dose dependent manner. (C and D) When IL-6 antibody or siRNA was added to A549 cells cultured with MSCs, sphere formation was significantly decreased, suggesting that IL-6 in MSCs was the main cytokine that promotes sphere formation in A549 and CL1-5 cells. (E) When A549 cells were cultured with 293T cells with an IL-6 stable clone, increased sphere formation was observed.
a mature, complex, physiological tumor microenvironment. They also pointed out that tumor associated fibroblasts derived from MSCs in the tumor microenvironment biologically impact tumor progression through the production of growth factors, cytokines, chemokines, matrix-degrading enzymes, and immune-modulatory
mechanisms [33]. In this study, we showed that when MSCs were cultured with lung cancer cells, increased sphere formation and overexpression of pluripotency markers including Nanog, Oct4A, and Sox2 were seen in lung cancer cells. Resistance to traditional chemotherapy of lung cancer was also seen in A549 lung cancer
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Fig. 5. A549 cells added with IL6 showed increased expression of pluripotency MSC markers and chemoresistance. (A) Western blotting analysis showed increased expression of pluripotency MSC markers including Nanog, Sox2, and Oct4A in A549 cells added with IL6, as compared to A549 cells alone. (B) Immunofluorescence study revealed expression of pluripotency MSC markers in A549 cells when IL6 was added. (C) DNA fragmentation assayed by the TUNEL technique revealed that apoptosis induced by a combination of cisplatin and gemcitabine treatment was also significantly reduced in A549 cells mixed with IL6, as compared to A549 cells alone.
cells cultured with MSCs. WI38, human lung fibroblast cells, were used in the study for a control experiment to demonstrate the enhancement of sphere formation by MSCs is more obvious and specific in MSCs rather than other tissues derived fibroblasts. We found that compared to MSCs, WI38 can only slightly increase the sphere formation of A549 and CL1-5 lung cancer cells at a concentration of 9 × 104 , while MSCs can significantly increase the sphere formation of cancer cells at a concentration of less than 1 × 104 . The exact mechanism about why WI38 can only slightly increased the sphere formation of lung cancer cells is not clear. We further demonstrated that blockade of IL-6 secreted from MSCs and the downstream pathway proteins, including JAK2 and STAT3, decreased sphere formation in A549 and CL1-1 cells, as well as the capability of tumor formation, compared to when A549 cells were indirectly cultured with MSCs, indicating that IL-6 is an important cytokine secreted from MSCs, and can enhance the tumor
initiation of A549 and CL1-5 lung cancer cells in immunodeficient mice. IL-6 has been reported to be associated with some epithelial cancers including breast, colon, and lung [14–16]. Increased levels of IL-6 have been observed in a variety of different tumors, and predict an adverse outcome [12]. As to the mechanisms of IL-6 involvement in cancer, in 2007 Sansone et al. [13] reported that IL-6 induces malignant features in Notch3-expressing stem cells from human ductal breast carcinoma and the normal mammary gland. Gao et al. [17] showed that mutant epidermal growth factor receptors (EGFRs) could activate the gp130/JAK/STAT3 pathway by means of IL-6 upregulation in primary human adenocarcinoma. In 2009, Colomiere et al. [20] reported that activation of STAT3 in high-grade ovarian carcinomas may occur directly through activation of EGFR or IL-6R, or indirectly through induction of IL-6R signaling. The results of our study suggest that MSCs from a tumor
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Fig. 6. Sphere formation of A549 and CL1-5 cells decreased when the IL6/JAK2/STAT3 pathway was inactivated. (A) Western blotting analysis showed that IL-6 activated phosphorylated JAK2 and STAT3 in A549 cells. (B) Western blotting analysis demonstrated that ag490 inhibited phosphorylated JAK2 and STAT3. (C) Sphere formation was decreased when ag490 was added to A549 and CL1-5 cells. STAT3 siRNAs and STAT3 expression was inactivated and sphere formation decreased in A549 and CL1-5 cells cultured with MSCs.
microenvironment may secret IL-6 to enhance tumor initiation in lung cancer through activation of the IL-6/JAK2/STAT3 pathway. Whether MSCs are involved in tumor development by secreting cytokines, or that MSCs are recruited to the microenvironment to facilitate the tumor progression is undetermined. It has been reported that the stroma has an active, oncogenic role in tumorigenesis [34,35]. In 2005, Orimo et al. [36] reported that carcinoma-associated fibroblasts extracted from human breast carcinoma promote the growth of breast carcinoma cells. In 2008,
Mishra et al. [37] reported that MSCs become activated and resemble carcinoma-associated myofibroblasts on prolonged exposure to condition medium from breast cancer cells. Contrary to our findings, Rattigan et al. [38] recently demonstrated that under hypoxic conditions, breast cancer cells secret high levels of IL-6, which serve to activate and attract MSCs. Further study to elucidate the interplay between MSCs and the tumor microenvironment is important. In summary, our data showed that when A549 or CL1-5 lung cancer cells were cultured with MSCs, increased sphere forma-
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Fig. 7. Reduced capability of tumor formation of MSCs in A549 lung cancer cells when IL-6 was inhibited in MSCs or STAT3 was silenced in A549 cells mixed with MSCs. (A) Immunohistochemistry studies of tumor blocks retrieved from the animals showed that increased protein expression of phosphorylated JAK2 and STAT3 was observed in tumors formed by A549 cells mixed with MSCs, compared to tumors formed by A549 cells alone. (B) Tumors were formed in mice injected with A549 cells mixed with MSCs. However, when IL-6 siRNA or STAT3 siRNA was added to the A549 cells mixed with MSCs, the capacity of MSCs to form tumors was blocked, suggesting that the capacity of MSCs to promote tumor formation is through activation of the IL-6/JAK2/STAT3 pathway. (C) Tumor formation in immunodeficient mice when CL1-5 lung cancer cells were injected in the study, showing the same results as when A549 cells were used, although CL1-5 cells were less capable of forming tumors as compared to A549 cells.
tion, drug resistance and overexpression of pluripotency markers were observed, as compared to A549 or CL1-5 cells cultured alone, through activation of the IL-6/JAK2/STAT3 pathway. In in vivo study, MSCs can enhance the capability of A549 and CL1-5 lung cancer cells to form tumors in immunodeficient mice. Upon blockade of the IL-6/JAK2/STAT3 pathway, the capability of A549 and CL1-5 lung cancer cells to form tumors was attenuated. Further investigation to study the role of MSCs and the secretion of cytokines in lung cancer tumorigenesis is mandatory. Conflict of interest The authors declare no conflicts of interest.
Acknowledgements This work was assisted in part by the Division of Experimental Surgery of the Department of Surgery, Taipei Veterans General Hospital, and supported by the Grant V99C1-035 from Taipei Veterans General Hospital to Han-Shui Hsu and the Lung Cancer Foundation in Memory of Doctor KS Lu. References [1] Tlsty TD. Stromal cells can contribute oncogenic signals. Semin Cancer Biol 2001;11:97–104. [2] Elenbaas B, Weinberg RA. Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation. Exp Cell Res 2001;264:169–84.
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