Accepted Manuscript Title: Mesenchymal stem cells in lung cancer tumor microenvironment: their biological properties, influence on tumor growth and therapeutic implications Author: Renwang Liu, Sen Wei, Jun Chen, Song XU PII: DOI: Reference:

S0304-3835(14)00426-1 http://dx.doi.org/doi:10.1016/j.canlet.2014.07.047 CAN 11970

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Cancer Letters

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18-5-2014 10-7-2014 30-7-2014

Please cite this article as: Renwang Liu, Sen Wei, Jun Chen, Song XU, Mesenchymal stem cells in lung cancer tumor microenvironment: their biological properties, influence on tumor growth and therapeutic implications, Cancer Letters (2014), http://dx.doi.org/doi:10.1016/j.canlet.2014.07.047. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Mesenchymal Stem Cells in lung cancer tumor microenvironment: their biological properties, influence on tumor growth and therapeutic implications

Renwang Liu1*, Sen Wei1*, Jun Chen1,2#, Song XU1,2#

1

Department of Lung Cancer Surgery, 2Tianjin key laboratory of lung cancer metastasis and tumor microenvironment, Lung Cancer Institute, Tianjin Medical University General Hospital, 300052, Tianjin, China To whom correspondence may be addressed: Song XU, MD, PhD, Department of Lung Cancer Surgery, Lung Cancer Institute, Tianjin Medical University General Hospital, No.154 Anshan Road, HepingDistritct, 300052, Tianjin, China. E-mail: [email protected]; Tel: +86 22 60817272; Fax: +86 22 60363013 Jun Chen, MD, PhD, Department of Lung Cancer Surgery, Lung Cancer Institute, Tianjin Medical University General Hospital, No.154 Anshan Road, HepingDistritct,300052, Tianjin, China; E-mail:[email protected];Tel: +86 22 60814803; Fax: +86 22 60363013 *These authors contribute equally to this paper. Running title: MSCs in lung cancer tumor microenvironment

Highlights

 MSCs in lung cancer represent genetic and phenotypic abnormalities.  MSCs influence the growth, metastasis and drug resistance of lung cancer.  MSCs act as a potential therapeutic approach of lung cancer.

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Abstract The tumor microenvironment （TME）of lung cancer has been documented to play an important role in participating in tumor disease progression. As the precursor of most stroma in TME, mesenchymal stem cells (MSCs) draw great attention since evidence has suggested that MSCs derived from lung cancer patients present a different phenotype compared to their normal counterparts. Furthermore, MSCs could be recruited towards tumor sites and influence tumor survival, although the effect remains contradictory. Our review will summarize the current advance of the role MSCs in lung cancer and explore the possible treatment strategies by blocking their crosstalk.

Keywords: mesenchymal stem cells, lung cancer, microenvironment.

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1. Introduction Lung cancer is the most common cancer in terms of both incidence and mortality worldwide. Although significant progress has been made in the understanding of disease pathogenesis and development of novel therapies, lung cancer remains an incurable disease. The main types of lung cancer are small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). The initiation and development of lung cancer ascribe to genetic and certain microenvironmental factors, including exposure to tobacco smoke and air pollution. It has documented well that the stromal cells, growth factors, cytokines/chemokines within tumor surroundings constitute a microenvironment favoring the proliferation, survival, migration and drug resistance of lung cancer cells. As the adult stem cells with the capacity of differentiation towards a variety of cell types, mesenchymal stem cells (MSCs) were traditionally found in the bone marrow (BM), but can also be isolated from other tissues including cord blood, peripheral blood, fallopian tube, fetal liver and lung. Advancements in MSC research have shed light on how these stem cells are used in various clinical applications, including immunomodulatory therapies (i.e., prevention of graft-versus-host disease or treatment of Crohn’s disease) and cell replacement therapies for mesenchymal tissues such as bone and cartilage. Previous studies have demonstrated that, as the precursors of most stromal cells, MSCs are involved in the disease progression of different tumor types, including lung cancer. In addition, lung cancer patient-derived MSCs exhibited different differentiation potentials and phenotypic characterization. Exploration of 3 Page 3 of 23

the interaction between lung cancer and surrounding stromal cells, in special for MSCs, will be helpful for an in-depth understanding the lung cancer disease progression. Here, we review the update advance of the biological properties of MSCs derived from lung cancer disease, and their possible role in the biology and treatment of lung cancer.

2. The abnormality of MSCs in lung cancer Some evidence has demonstrated that MSCs derived from individuals under pathological condition, exhibit genetic and functional abnormalities compared to their normal counterpart [1-4]. Similar to other cancers, the abnormality of MSCs was also been reported in lung cancer recently. Gottschling et al. performed a comparative analysis of MSCs from lung cancer tissue and homologous normal lung tissue of 15 patients on the level of molecular and functional properties [5]. The results revealed that MSCs in lung cancer tissue showed accelerated growth kinetics, reduced sensitivity to cisplatin and differential expression of butyrylcholinesterase, clusterin and quiescin Q6 sulfhydryl oxidase 1 compared to normal MSCs. Fernándezet et al. found that MSCs from BM of lung cancer patients without bone metastasis presented low capacity of osteogenic and adipogenic differentiation and increased chondrogenic differentiation [6]. And Hofer et al. demonstrated that BM derived MSCs of lung cancer patients exhibited decreased expression of platelet-derived growth factor receptor (PDGFR)-alpha, transforming growth factor receptor (TGFbetaR)-I, II, III, epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR)-I, c-Fos and c-Myc compared to those from healthy donors [7]. Why MSCs exhibit abnormal in lung cancer? The exact mechanism is still not clear. According to our previous experience, multiple myeloma (MM) cells could trigger MSCs upregulation miR-135b expression which decreased MSCs osteogenic differentiation and induced MM bone disease, and miR-135b expression returned to normal level after the removal of MM cells [8]. The abnormality of MSC is transient due to the short coculture in vitro with tumor cells. However, during the long-term exposure to the MM tumor microenvironment in vivo, the mRNA and/or miRNA profile of MSCs might have undergone irreversible changes. This might explain in part how MSCs gain phenotypic abnormality in lung cancer as well. 4 Page 4 of 23

Therefore, since MSCs in lung cancer represented genetic and phenotypic abnormalities, one of the interesting topics deserved for further investigation is to identify the function of these abnormalities in lung cancer regarding to tumor growth and progression, and to exploit novel treatment modalities by correcting or targeting these abnormalities.

3. Effects of MSCs on lung cancer growth and progression Tumors are composed of neoplastic compartment (“seed”) and non-neoplastic tissue (“soil”). These non-neoplastic tissue, including extracellular matrix, immune and inflammatory cells, connective tissue, and other mesenchymal components, are not just by-standers but also involved in the progression of cancer. Recent studies showed that MSCs, as the precursor of most stromal cells, contribute to construct the tumor-associated stroma and influence tumor growth. However, the effect of MSCs on proliferation and survival of lung cancer cells is contradictory according to recent publications (Fig. 1). 3.1 MSCs favor the growth of lung cancer Accumulating evidence suggests that MSCs show the ability to protect lung cancer cells from apoptosis and promote tumor growth. A549 lung cancer cells pretreated with starvation were co-cultured with MSCs in transwell system, resulting in a higher viability and apoptosis reduction [9]. MSCs also increased angiogenesis and promoted C57BL/6 mice lung cancer growth when co-injected with MSCs and Lewis lung carcinoma (LLC) cells in vivo [10]. In addition, MSCs could reduce the apoptosis of lung cancer cells via up-regulating stanniocalcin-1 (STC1) [11], which leaded to enhanced Warburg effect and reduced intracellular reactive oxygen species (ROS) [12]. Adipose tissue is a rich source of multipotent MSCs, which are thought to be functionally similar to bone marrow-derived MSCs. In terms of adipose tissue-derived mesenchymal stem cells (ASCs), the growth of lung cancer was also been promoted via co-injection with ASCs in vivo [13]. And it was reported that tumorigenesis and angiogenesis of lung cancer were facilitated when co-injected with ASCs and tumor cells into mice [14]. 3.2 MSCs inhibit lung cancer progression On the other hand, there were also several studies reporting the contradictory results that MSCs inhibited lung cancer tumor progression. Tian et al. showed that MSCs could inhibited 5 Page 5 of 23

the proliferation and induced the apoptosis of A549 cells, probably through downregulating the expressions of proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (Bcl-2) and inhibiting the formation of Cyclin E-cyclin-dependent kinase 2 (CDK2) complexes [15]. A Chinese group also confirmed that hMSCs could inhibit the proliferation and induce apoptosis of A549 as well as SK-MES-1 cells [16]. They proposed that MSCs-conditioned medium could downregulate the expression of VEGF in the tumor tissue to suppress angiongenesis and hence inhibited lung tumor growth. Moreover, MSCs also suppressed the growth of lung metastasis from breast cancer cells and resulted in a delay of in tumor progression according to Keramidas et al’s study [17].

3.3 Mechanisms of MSC mediated tumor growth and suppression Angiogenesis support Angiogenesis is a complex process and plays a significant role in tumor growth. It was demonstrated that there were more vessel area in the tumor sites co-injected with MSCs compared to tumor cells injection alone, indicating that MSCs may promote tumorigenesis via the enhancement of neovascularization [10]. However, in what appears to be a direct conflict with this data, MSCs inhibit capillary growth under certain conditions. Li et al. found that BM-MSCs derived conditioned medium (CM) could downregulate the expression of vascular endothelial growth factor (VEGF) in the tumor tissue to suppress angiongenesis and hence inhibiting lung tumor growth [16]. Carcinoma-associated fibroblasts Evidence has shown that lung cancer cells may stimulate MSCs to differentiate into carcinoma-associated fibroblasts (CAFs) which in turn enhance proliferation and tumor growth of lung cancer[14, 18]. Do et al. found that lung cancer cell A549 CM induced ASCs to express a disintegrin and metalloprotease 12 (ADAM12) via the LPA/LPA receptor 1 signaling axis [19]. ADAM12, belonging to the family of adhesion proteins and metalloproteases, is markedly overexpressed and acts as a prognostic marker in lung cancer [20, 21]. They further demonstrated that ADAM12 probably could stimulate the differentiation of ASCs to CAFs [19]. Additionally, LPA, as a small bioactive phospholipid, was also essential in the lung cancer cell induced expression of ADAM12 [22, 23]. ASCs, 6 Page 6 of 23

which were pretreated with LPA receptor 1 inhibitor Ki16425, resulted in the abrogation of ADAM12 expression according to Do’s study [19]. Thus, lung cancer may enhance self-proliferation via the LPA-stimulated ADAM12 expression and stimulate differentiation of CAFs from MSCs. Study methods Although several mechanisms of the effect of MSC on lung cancer were stated above,

the differences in the methodology of these reported studies may influence the contradictory findings as well. The time of MSCs introduction into tumors might be a

critical factor explaining the contradicting results. Other possible explanations for these conflicting outcomes might include the ratio of MSC numbers to cancer cell numbers that were used in the different studies, as well as the nature of the different tumor cell lines and animal models. The detail will be discussed below and listed in Table 1.

4. MSCs in lung cancer distant metastasis Lung cancer has often spread beyond the initial tumor at the time of diagnosis, due to the sufficient ability of its primary tumor cells to survive at distant metastatic sites [24]. During the metastasis progress, disseminated cells from primary lung tumor require self-renewal capability to survive in the distant tumor microenvironment [25]. Recent study revealed that MSCs could hybridize with NSCLC cells spontaneously which contributed to the acquisition of EMT and stem-cell like properties for cancer cells [26]. This enhances the self-renewal capability of the disseminated tumor cells via the up-regulation of vimentin, α-SMA and fibronectin, and down-regulation of E-cadherin and pancytokeratin. On the other hand, as discussed above, MSCs were capable of differentiating into CAFs in lung cancer [14]. Evidence has shown that CAFs could also enhance the metastatic potential of NSCLC via inducing EMT, up-regulating the expression of α-SMA, fibroblast activation protein (FAP), SMAD3 and activating the hedgehog signaling pathway [27].Given to these properties, MSCs may promote the distant metastasis of lung cancer (Fig. 2).

5. MSCs in the drug resistance of lung cancer 7 Page 7 of 23

Tumor microenvironment factors, including growth factors, cytokines, cell-cell and cell-matrix adhesion molecules and hypoxia, confer tumor cells resistance to chemotherapy. Many studies have reported that MSCs participated in the construction of a favorable condition that contributes to drug resistance for cancer cells [28, 29]. MSCs exert their effects through directly promoting resistance effectors and/or indirectly modulating other environmental factors. Regarding the effect of MSCs on the drug resistance of lung cancer, the reports are relatively few. Bergfeld et al. found that MSCs could strongly be attracted by lung cancer cells and decrease significantly the apoptosis of tumor cells induced by paclitaxel or doxorubicin, suggesting that MSCs may play a crucial role in the chemotherapeutic resistance of lung cancer [30]. It has also demonstrated that MSCs can alleviate the effect of cisplatin on LLC cells [31]. In addition, there was evidence showing that CAFs contributed to lung cancer cells resistance to the EGFR tyrosine kinase inhibitor (EGFR-TKI) gefitinib [32]. Since MSCs are capable of differentiating to CAFs [14], we speculate that MSCs might diminish the effectiveness of EGFR-TKI on lung cancer as well.

6. MSCs act as a potential therapeutic approach of lung cancer 6.1 MSCs act as a delivery vehicle for the therapy of primary and metastatic lung tumor Evidence has demonstrated that MSCs are capable of homing and delivering anti-tumor agents as a gene therapy vehicle to the tumor sites in certain cancer types, including lung cancer (Fig. 3). A recent study showed that human umbilical cord-derived MSCs (UC-MSCs) transduced with interleukin-24 (IL-24) exerted an anti-angiogenic effect and increased the expression of p21 and the cleaved caspases-3/8/9 in A549 cells, resulting in the suppression of lung cancer growth significantly in vitro and in vivo [33]. Using a mouse xenograft model, Stoff-Khalili et al. demonstrated that conditionally replicating adenoviruses (CRAd) trasnsduced MSCs significantly inhibited the growth of MDA-MB-231 breast cancer cells pulmonary metastasis and prolonged the mouse survival compared to mice treated with CRAd alone [34]. MSCs genetically modified with pigment epithelium-derived factor (PEDF) and oncolytic adenoviruses also showed inhibitory effects on the growth of lung tumors in vitro and in vivo[35, 36]. In addition, PE (ΔIII)-E7-KDEL3 (a protein vaccine that can induce immunity against cervical cancer specifically) [37], NK4 (an antagonist of hepatocyte growth 8 Page 8 of 23

factor) [38], and CX3CL1 (an immunostimulatory chemokine) [39] have been used to reconstruct MSCs for gene therapy which were introduced into lung metastatic tumor tissues. MSC-based gene therapy has exhibited an effective anti-cancer strategy compared to using these biological agents alone. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), as a member of TNF family [40], is able to induce tumor-selective apoptosis in many cancers [41, 42]. Nevertheless, the expression of TRAIL is down-regulated in metastatic prostate, colon, and breast cancer [43], suggesting that TRAIL inducing apoptosis may be inhibited in the metastatic cancers. However, study showed that hMSCs pre-activated with TNF-α can enhance the effect of TRAIL on the pulmonary metastasis of breast cancer and trigger the apoptosis of MDA-MB-231 breast cancer cells [44]. In addition, TRAIL-expressing MSCs also decreased the growth of primary tumor of lung cancer as well as lung metastasis from breast cancer in mice [45-48]. Furthermore, several other molecules could be exploited to reconstruct MSCs in terms of gene therapy for lung primary and metastatic tumors. Ren et al. demonstrated that MSCs producing interferon (IFN)-alpha could reduce the growth of lung metastasis from melanoma using a B16F10 melanoma mice model [49]. Matsuzuka et al. showed that hUCMSCs transfected with IFN-beta (IFN-beta-hUCMSCs) can suppress the proliferation of lung adenocarcinoma cell line H358 in vitro and tumor growth in vivo [50]. Moreover, MSCs expressing interleukin-12 (MSCs/IL-12M) showed the capability of sustained expressions of IL-12 and IFN-γ and significantly inhibited lung metastasis in B16F10 melanoma mice model [51].

6.2 MSCs contribute to alleviate radio- and chemo- therapy induced side effects Radiotherapy plays an important role in the treatment of lung cancer and has been strongly recommended especially for the advanced lung cancer patients by NCCN guideline. However, it is clear that radiation therapy can cause lung injury including radiation pneumonitis and pulmonary fibrosis [52]. Intriguingly, MSCs, due to their capability of homing to radiation-injured tissue [53], may provide a potential therapeutic regimen to alleviate this radiotherapy side effect in lung cancer patients. It was demonstrated that MSCs 9 Page 9 of 23

could protect the alveolar type II (ATII) cells from apoptosis induced by radiation in vitro, and moreover were capable of migrating towards the radiation-induced lung injury (RILI) and decreasing the mortality caused by RILI in mice in vivo [54, 55]. Therefore, MSCs may contribute to the treatment effects of radiotherapy in lung cancer via protecting lung tissue from radiation injury. Furthermore, study showed that fetal human umbilical cord perivascular cells (HUCPCs), identified as a kind of MSCs, could engraft bleomycin (BLM) damaged ATII [56]. Ortiz et al. demonstrated that MSCs could home to injured lung tissue by BLM and reduce the inflammation and collagen deposition [57]. Moreover, Di et al. proposed that MSCs could repair the cardiomyopathy and decrease the apoptosis in intestinal crypts associated with adriamycin (ADM) [58]. Thus, we can draw a conclusion that MSCs may exhibit the ability to alleviate the side effects induced by chemo- and radio-therapy in lung cancer disease.

7. Conclusion and perspectives Lung cancer remains an incurable disease and the most common approaches for lung cancer treatment are chemo- and radio-therapy besides surgical resection. MSCs play a crucial role in participating in lung cancer cell growth, metastasis and drug resistance, and may provide a useful therapeutic approach of this incurable disease. However, the interaction between MSCs and lung cancer is poorly understood and the effect of MSCs on lung cancer growth and progression is still controversial. According to the studies presented above, we speculate that the contradictory results may due to ratio of MSCs and tumor cells, source of MSCs and MSCs injection time and frequency. Intriguingly, all of the in vitro experiments that co-culture MSCs and lung cancer cells with the ratio of 1:1 were presented the results of inhibition of lung cancer cells proliferation. On the contrary, the opposite results came from the ratio of 1:2 and 1:10 (MSCs: lung cancer cells) respectively, which tumor cells are the majority in the coculture system. Hence, we propose that a high population of MSCs in the tumor microenvironment may inhibit the proliferation of lung cancer cells. Moreover, we observe that in many studies with directly coculture of MSCs and lung cancer cells, MSCs show a favorable effect on tumor growth, while in other studies lung cancer cells exhibit decreased growth in the exposure of MSCs derived conditioned medium. This indicates that 10 Page 10 of 23

MSCs may exert different effects on lung cancer growth via paracrine and cell-cell contact mechanism, respectively. Further studies have to figure out the underlying mechanisms of the dual effects of MSCs on the growth of lung cancer. MSCs derived from lung cancer patients exhibit abnormal phenotypes compared to their normal counterparts, including accelerated growth kinetics, decreased expression of PDGFR-alpha, TGFbetaR-I, II, III, EGFR, fFGFR-I, c-Fos and c-Myc. Further identifying the mechanisms and function of these abnormalities may exploit the novel treatment approaches by correcting or targeting these abnormalities. On the other hand, MSCs are capable of homing and delivering anti-tumor agents including IL-12, IL-24, CRAd, PE(ΔIII)-E7-KDEL3, NK4, CX3CL1, PEDF, oncolytic adenoviruses, IFN-α, -β and –γ as gene therapy vehicle to the primary and metastatic lung tumor sites[59]. Future studies have to pay attention to the exploration of more effective and safe anti-tumor agents for the clinical application. Moreover, MSCs can alleviate side effect of radio- and chemo-therapy by repairing the treatment induced tissue injury, however, the treatment efficiency is not influenced. This may provide the basis for the clinical practice of improving the treatment effects of conventional regimen in lung cancer disease. Moreover, besides MSCs, other tumor stromal components, such as macrophage, adipocytes, and ECM, are reported to be involved in the tumor progression. All of these non-cancerous populations construct together a tumor microenvironment which favors tumor cell growth, inhibits apoptosis, and induces drug resistance. Exploration of the interaction of MSCs and lung cancer cells will be a good study model which contributes to the understanding the crosstalk between cancer cells and other stromal components. In conclusion, MSCs, as the precursors of most stromal cells, has been documented to be involved in lung cancer growth, metastasis and drug resistance. MSCs from lung cancer patients present a different genetic and functional characterization compared to those from healthy donors. Investigations of the interaction between MSCs and lung cancer cells may provide a model to further research the roles of other stromal cells in lung cancer tumor microenvironment. However, the effects of MSCs on the growth, metastasis and drug resistance of lung cancer remain indeterminate and the mechanism of MSCs act as a delivery vehicle for the therapy is still indistinct. Further studies are required to investigate the role of 11 Page 11 of 23

MSCs in lung cancer tumor microenvironment, especially in the issues of tumor growth, metastasis, drug resistance, and therapy.

Acknowledgements This work was supported by grants from National Natural Science Foundation of China (81301812,81172233), Specialized Research Fund for the Doctoral Program of Higher Education (20131202120004), Scientific Research Foundation for the Returned Overseas Chinese Scholars of State Education Ministry, Tianjin Educational Committee Foundation (20120117) and Wu Jieping Medical Foundation (320.6750.1377, 320.6750.1376, 320.6750.12341). Conflict of Interest The authors declare that there are no conflicts of interest.

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[34] M.A. Stoff-Khalili, A.A. Rivera, J.M. Mathis, N.S. Banerjee, A.S. Moon, A. Hess, R.P. Rocconi, T.M. Numnum, M. Everts, L.T. Chow, J.T. Douglas, G.P. Siegal, Z.B. Zhu, H.G. Bender, P. Dall, A. Stoff, L. Pereboeva, D.T. Curiel, Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma, Breast Cancer Res Treat, 105 (2007) 157-167. [35] Q. Chen, P. Cheng, T. Yin, H. He, L. Yang, Y. Wei, X. Chen, Therapeutic potential of bone marrow-derived mesenchymal stem cells producing pigment epithelium-derived factor in lung carcinoma, Int J Mol Med, 30 (2012) 527-534. [36] T. Hakkarainen, M. Sarkioja, P. Lehenkari, S. Miettinen, T. Ylikomi, R. Suuronen, R.A. Desmond, A. Kanerva, A. Hemminki, Human mesenchymal stem cells lack tumor tropism but enhance the antitumor activity of oncolytic adenoviruses in orthotopic lung and breast tumors, Hum Gene Ther, 18 (2007) 627-641. [37] H.J. Wei, A.T. Wu, C.H. Hsu, Y.P. Lin, W.F. Cheng, C.H. Su, W.T. Chiu, J. Whang-Peng, F.L. Douglas, W.P. Deng, The development of a novel cancer immunotherapeutic platform using tumor-targeting mesenchymal stem cells and a protein vaccine, Mol Ther, 19 (2011) 2249-2257. [38] M. Kanehira, H. Xin, K. Hoshino, M. Maemondo, H. Mizuguchi, T. Hayakawa, K. Matsumoto, T. Nakamura, T. Nukiwa, Y. Saijo, Targeted delivery of NK4 to multiple lung tumors by bone marrow-derived mesenchymal stem cells, Cancer Gene Ther, 14 (2007) 894-903. [39] H. Xin, M. Kanehira, H. Mizuguchi, T. Hayakawa, T. Kikuchi, T. Nukiwa, Y. Saijo, Targeted delivery of CX3CL1 to multiple lung tumors by mesenchymal stem cells, Stem Cells, 25 (2007) 1618-1626. [40] S.R. Wiley, K. Schooley, P.J. Smolak, W.S. Din, C.P. Huang, J.K. Nicholl, G.R. Sutherland, T.D. Smith, C. Rauch, C.A. Smith, et al., Identification and characterization of a new member of the TNF family that induces apoptosis, Immunity, 3 (1995) 673-682. [41] H. Walczak, M.A. Degli-Esposti, R.S. Johnson, P.J. Smolak, J.Y. Waugh, N. Boiani, M.S. Timour, M.J. Gerhart, K.A. Schooley, C.A. Smith, R.G. Goodwin, C.T. Rauch, TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL, EMBO J, 16 (1997) 5386-5397. [42] F.A. Kruyt, TRAIL and cancer therapy, Cancer Lett, 263 (2008) 14-25. [43] J.E. Allen, W.S. El-Deiry, Regulation of the human TRAIL gene, Cancer biology & therapy, 13 (2012) 1143-1151. [44] R.H. Lee, N. Yoon, J.C. Reneau, D.J. Prockop, Preactivation of human MSCs with TNF-alpha enhances tumor-suppressive activity, Cell Stem Cell, 11 (2012) 825-835. [45] M.R. Reagan, F.P. Seib, D.W. McMillin, E.K. Sage, C.S. Mitsiades, S.M. Janes, I.M. Ghobrial, D.L. Kaplan, Stem Cell Implants for Cancer Therapy: TRAIL-Expressing Mesenchymal Stem Cells Target Cancer Cells In Situ, J Breast Cancer, 15 (2012) 273-282. [46] A. Mohr, M. Lyons, L. Deedigan, T. Harte, G. Shaw, L. Howard, F. Barry, T. O'Brien, R. Zwacka, Mesenchymal stem cells expressing TRAIL lead to tumour growth inhibition in an experimental lung cancer model, J Cell Mol Med, 12 (2008) 2628-2643. [47] Y.L. Hu, B. Huang, T.Y. Zhang, P.H. Miao, G.P. Tang, Y. Tabata, J.Q. Gao, Mesenchymal stem cells as a novel carrier for targeted delivery of gene in cancer therapy based on nonviral transfection, Mol Pharm, 9 (2012) 2698-2709. [48] M.R. Loebinger, A. Eddaoudi, D. Davies, S.M. Janes, Mesenchymal stem cell delivery 15 Page 15 of 23

of TRAIL can eliminate metastatic cancer, Cancer Res, 69 (2009) 4134-4142. [49] C. Ren, S. Kumar, D. Chanda, J. Chen, J.D. Mountz, S. Ponnazhagan, Therapeutic potential of mesenchymal stem cells producing interferon-alpha in a mouse melanoma lung metastasis model, Stem Cells, 26 (2008) 2332-2338. [50] T. Matsuzuka, R.S. Rachakatla, C. Doi, D.K. Maurya, N. Ohta, A. Kawabata, M.M. Pyle, L. Pickel, J. Reischman, F. Marini, D. Troyer, M. Tamura, Human umbilical cord matrix-derived stem cells expressing interferon-beta gene significantly attenuate bronchioloalveolar carcinoma xenografts in SCID mice, Lung Cancer, 70 (2010) 28-36. [51] S.H. Seo, K.S. Kim, S.H. Park, Y.S. Suh, S.J. Kim, S.S. Jeun, Y.C. Sung, The effects of mesenchymal stem cells injected via different routes on modified IL-12-mediated antitumor activity, Gene Ther, 18 (2011) 488-495. [52] F. Cappuccini, T. Eldh, D. Bruder, M. Gereke, H. Jastrow, K. Schulze-Osthoff, U. Fischer, D. Kohler, M. Stuschke, V. Jendrossek, New insights into the molecular pathology of radiation-induced pneumopathy, Radiother Oncol, 101 (2011) 86-92. [53] M. Mouiseddine, S. Francois, A. Semont, A. Sache, B. Allenet, N. Mathieu, J. Frick, D. Thierry, A. Chapel, Human mesenchymal stem cells home specifically to radiation-injured tissues in a non-obese diabetes/severe combined immunodeficiency mouse model, Br J Radiol, 80 Spec No 1 (2007) S49-55. [54] J. Xue, X. Li, Y. Lu, L. Gan, L. Zhou, Y. Wang, J. Lan, S. Liu, L. Sun, L. Jia, X. Mo, J. Li, Gene-modified mesenchymal stem cells protect against radiation-induced lung injury, Mol Ther, 21 (2013) 456-465. [55] L.V. Kursova, A.G. Konoplyannikov, V.V. Pasov, I.N. Ivanova, M.V. Poluektova, O.A. Konoplyannikova, Possibilities for the use of autologous mesenchymal stem cells in the therapy of radiation-induced lung injuries, Bull Exp Biol Med, 147 (2009) 542-546. [56] T. Montemurro, G. Andriolo, E. Montelatici, G. Weissmann, M. Crisan, M.R. Colnaghi, P. Rebulla, F. Mosca, B. Peault, L. Lazzari, Differentiation and migration properties of human foetal umbilical cord perivascular cells: potential for lung repair, J Cell Mol Med, 15 (2011) 796-808. [57] L.A. Ortiz, F. Gambelli, C. McBride, D. Gaupp, M. Baddoo, N. Kaminski, D.G. Phinney, Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects, Proc Natl Acad Sci U S A, 100 (2003) 8407-8411. [58] G.H. Di, S. Jiang, F.Q. Li, J.Z. Sun, C.T. Wu, X. Hu, H.F. Duan, Human umbilical cord mesenchymal stromal cells mitigate chemotherapy-associated tissue injury in a pre-clinical mouse model, Cytotherapy, 14 (2012) 412-422. [59] L.J. Dai, M.R. Moniri, Z.R. Zeng, J.X. Zhou, J. Rayat, G.L. Warnock, Potential implications of mesenchymal stem cells in cancer therapy, Cancer Lett, 305 (2011) 8-20.

16 Page 16 of 23

Figure legends Figure 1: Schematic model for the dual effect of MSCs on the growth of lung cancer MSCs play a dual effect on the growth of lung cancer. Report shows that BM-MSCs could favor the lung cancer (LC) cells growth via activating autophagy and up-regulating stanniocalcin-1(STC-1) which result in inhibition of tumor apoptosis, enhancing the Warburg effect and stimulating the angiogenesis. On the other hand, bone marrow-derived mesenchymal stromal cells (BM-MSCs) conditioned medium (CM) could inhibit tumor growth by down-regulating PCNA, Bcl-2, Cyclin E, pRB and VEGF expression in LC cells. Furthermore, regarding adipose tissue-derived mesenchymal stem cells (ASCs), LC cells CM could induce the differentiation of ASCs to carcinoma-associated fibroblasts (CAFs) through up-regulating LPA-stimulated ADAM12 expression and promote the growth of LC cells by increasing neovascularization. Figure 2: The mechanism of MSCs promote distant metastasis of lung cancer MSCs could hybridize with lung cancer (LC) cells spontaneously which contribute to the acquisition of epithelial-to-mesenchymal transition (EMT) and stem-cell like properties for cancer cells. This enhances the self-renewal capability of the disseminated tumor cells via the upregulation of vimentin, α-smooth muscle actin (α-SMA) and fibronectin, and down-regulation of E-cadherin and pancytokeratin. Carcinoma-associated fibroblasts (CAFs), which could differentiate from MSCs, could also enhance the metastatic potential of LC cells by inducing EMT, up-regulating the expression of α-SMA, fibroblast activation protein (FAP) and SMAD family number-3 and activating the Hedgehog signaling pathway. All of these factors could promote distant metastasis of lung cancer directly or indirectly. Figure 3: The therapeutic effect of MSCs on primary and metastatic lung tumor. MSCs presented a therapeutic potential of lung cancer according to recent studies. Anti-tumor agents were demonstrated effective in the treatment of lung tumor when delivered by MSCs via viral-transfection or nonviral-transfection. In the item of primary lung tumor, the agents 17 Page 17 of 23

include oncolytic adenoviruses, PEDF, IFN-beta and IL-24. And regarding metastatic lung tumor, CRAd, PE(ΔIII)-E7-KDEL3, NK4, CX3CL1, IFN-alpha and IL-12M were demonstrated as candidates for the treatment. Meanwhile, MSCs with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression could decrease the growth of both primary and metastatic lung tumor.

Table 1: Reports about MSC in lung tumor growth Author

Source of

Tumor

MSC: tumor cell ratio and

Tumor cell s

Findings

isolation

model

timing of infusion In vitro

In vivo

In vitro

In vivo

TIAN

Human

Lung

Six-week-

1:1, for 24 or

5 × 106 A549

MSC

MSC

et

BM

cancer cell

old female

48h

cells

single

inhibited

stimulated

derived

line A549

BALB/c

or

mixed

proliferatio

growth

nude mice

with 1 × 106

n

and

hMSCs

invasion,

increased

but

vessel

injected s.c.

promoted

formation

for one time

apoptosis

of tumors.

al.

[15]

MSCs

0.1ml

in PBS,

and

of A549. Human

Murine

Female

das

BM

mammary

athymic

TSA-pGL3

ation

derived

adenocarci

NMRI

cells

MSCs

MSCs

noma

nude mice

injection i.v.

decreased

line

aged 6 to

on

the growth

TSA-pGL3

8

MSCs

et

al. [17]

cell

weeks,

N/A

1.0

×

105

Kérami

day

0, i.v.

N/A

Administr

of

of

lung

18 Page 18 of 23

and

lung

injection on

metastatic

metastatic

day

tumors

model was

measured on

establishe

day 10

4,

d by cell line TSA-pGL 3 ZHAN

Human

Lung

Male

1:2, cultured

After

G

BM

carcinoma

NOD/SCI

for 6h, then

starvation，

derived

cell

D

switched

1.0

MSCs

A549

al. [9]

et

SPC-1

lines and

mice

aged weeks

4-6

to

24h

×

106

MSCs

hMSCs

activated

stimulated

autophagy,

the

DF-12 media

lung

decreased

initiation

without FBS

carcinoma

apoptosis

and

for 24 h

cells, single

and led to

growth of

or

better

lung

with 5.0 ×

tolerance

cancer

105 MSCs in

of

0.1ml PBS，

cancer

mixed

lung

injected s.c， cells under sacrificed on

serum-dep

day 30

rived conditions

19 Page 19 of 23

BLOC

Human

Lung

N/A

K et al.

BM

cancer cell

cultured

for

reduced

[11]

derived

line A549

24h,

and

apoptosis

MSCs

1:10,

N/A

MSCs

cocultures

of

were

cells,

incubated

increased

under

1%

N/A

A549

the

O2, 5% CO2

expression

and 94% N2

of

for 24h for

under

hypoxia

hypoxia at

experiment

pH 5.8 and

STC-1

restored inreacellua r STC-1 in A549 cells Suzuki

C57BL/6

Lewis lung

C57BL/6

et

mice BM

carcinoma

mice,

5:1, injected

increased

derived

(LLC) cells

6-8weeks

s.c.,

angiogene

of age

calculated

sis

every 2 days

promoted

after 5 days

tumor

[10]

al.

MSCs

N/A

0.2:1, 1:1 or

N/A

MSCs

and

20 Page 20 of 23

growth

Li et al.

Human

Lung

Six-week-

1:1,

Tumor cells

MSCs

Lung

[16]

BM

cancer cell

old female

co-cultures

treated with

inhibited

tumor

derived

line

BALB/c

for 48h ;

RPMI 1640

the

cells

MSCs

SK-MES-1

nude mice

Cancer cells

medium

proliferatio

treated

cultured

supplemente

n of lung

with

RPMI 1640

d with 10%

cancer

MSC-CM

medium

FBS and

cells

supplemente

MSCs-condit

MSC-CM

lower

d with 2%

ioned

promoted

incidence

FBS

medium

the

and

apoptosis

decreased tumor size

and A549

in

and

showed

the

MSCs-condit

(1:1)

ioned

MSC-growth

of

medium

medium

cancer

and

(MSC-CM)

(1:1) for 24h,

cells

downregul

(1:1)

injected s.c. ,

ated

MSC-growth

examined

VEGF

medium

every 5 days

expressio

or

or

and

lung

(MSC-GM)

sigificantl

(1:1)

y

21 Page 21 of 23

compared to

the

control group Jeon et

Human

Lung

BALB/c-n

al. [14]

adipose

carcinoma

u/nu mice

tissue-der

cell

ived

A549

1.0×106

N/A

N/A

hASCs

hASCs plus

stimulated

1.0×106

tumorigen

A549 cells in

esis

mesenchy

200 μl PBS,

angiogene

mal

s.c.

sis of lung

stem cells

examined

cancer

(hASCs)

twice

lines

and

weekly, sacrificed on week 4 Ohkou

Human

Lung

chi

BM

cancer cell

mentioned,

promoted

derived

line A549,

tumor

the

MSCs

H1299,

treated with

survival of

PC9, EBC1

100

lung

and LK2

H2O2

et

al. [12]

N/A

Ratio is not

induce

cell

μmol/l to

N/A

MSCs

N/A

cancer cells

22 Page 22 of 23

apoptosis,

except cell

and cultured

line

with

by

MSCs

LK2

or alone for

upregulati

7h, 48h and 5

ng

days

expression

the

of STC1 i.v.: intravenous; s.c.: subcutaneous

23 Page 23 of 23