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• W2160392015 abstract "Mesenchymal stem/stromal cells (MSCs) can either suppress or promote tumors. We found previously that incubation of human bone marrow MSCs (hMSCs) with TNF-α upregulated multiple genes including TRAIL, which has cancer apoptotic activity. Here, we show that weekly infusions into mice of hMSCs preactivated with TNF-α inhibited the progression of lung tumors formed from MDA-MB-231 breast cancer cells (MDA). In coculture, preactivated hMSCs induced apoptosis in MDA and several other TRAIL-sensitive cancer cell lines. TRAIL was further upregulated by apoptotic MDA cells in a TLR3-dependent manner; this feedforward cycle increased MDA cell apoptosis, and the chemotherapeutic drug doxorubicin had a synergistic effect. Also, activated hMSCs secreted DKK3 to suppress MDA cell cycling, leading to a decrease in β-catenin and cyclin D1/D3 and an increase in p21. Thus, culturing hMSCs with TNF-α enhances their tumor-suppressive properties and may represent a useful strategy to develop hMSC-based approaches for the treatment of cancer. Mesenchymal stem/stromal cells (MSCs) can either suppress or promote tumors. We found previously that incubation of human bone marrow MSCs (hMSCs) with TNF-α upregulated multiple genes including TRAIL, which has cancer apoptotic activity. Here, we show that weekly infusions into mice of hMSCs preactivated with TNF-α inhibited the progression of lung tumors formed from MDA-MB-231 breast cancer cells (MDA). In coculture, preactivated hMSCs induced apoptosis in MDA and several other TRAIL-sensitive cancer cell lines. TRAIL was further upregulated by apoptotic MDA cells in a TLR3-dependent manner; this feedforward cycle increased MDA cell apoptosis, and the chemotherapeutic drug doxorubicin had a synergistic effect. Also, activated hMSCs secreted DKK3 to suppress MDA cell cycling, leading to a decrease in β-catenin and cyclin D1/D3 and an increase in p21. Thus, culturing hMSCs with TNF-α enhances their tumor-suppressive properties and may represent a useful strategy to develop hMSC-based approaches for the treatment of cancer. Mesenchymal stem cells (MSCs) exposed to TNF-α activate TRAIL and DKK3 expression Activated MSCs decrease tumors in mice caused by a human breast cancer cell line Activated MSCs also induce apoptosis in several different cancer cell lines in culture TRAIL from MSCs causes apoptosis and DKK3 decreases cell cycling of cancer cells Nonhematopoietic progenitor cells derived from bone marrow, known as mesenchymal stem cells or multipotent stromal cells (MSCs), have been investigated for the treatment of cancers because they are able to preferentially home to tumors and incorporate into tumor stroma (Kanehira et al., 2007Kanehira M. Xin H. Hoshino K. Maemondo M. Mizuguchi H. Hayakawa T. Matsumoto K. Nakamura T. Nukiwa T. Saijo Y. Targeted delivery of NK4 to multiple lung tumors by bone marrow-derived mesenchymal stem cells.Cancer Gene Ther. 2007; 14: 894-903Crossref PubMed Scopus (131) Google Scholar; Kucerova et al., 2007Kucerova L. Altanerova V. Matuskova M. Tyciakova S. Altaner C. Adipose tissue-derived human mesenchymal stem cells mediated prodrug cancer gene therapy.Cancer Res. 2007; 67: 6304-6313Crossref PubMed Scopus (361) Google Scholar; Studeny et al., 2004Studeny M. Marini F.C. Dembinski J.L. Zompetta C. Cabreira-Hansen M. Bekele B.N. Champlin R.E. Andreeff M. Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents.J. Natl. Cancer Inst. 2004; 96: 1593-1603Crossref PubMed Scopus (696) Google Scholar; Xin et al., 2007Xin H. Kanehira M. Mizuguchi H. Hayakawa T. Kikuchi T. Nukiwa T. Saijo Y. Targeted delivery of CX3CL1 to multiple lung tumors by mesenchymal stem cells.Stem Cells. 2007; 25: 1618-1626Crossref PubMed Scopus (148) Google Scholar), but previous research has yielded conflicting results. Some reports showed that MSCs inhibited tumor growth (Djouad et al., 2006Djouad F. Bony C. Apparailly F. Louis-Plence P. Jorgensen C. Noël D. Earlier onset of syngeneic tumors in the presence of mesenchymal stem cells.Transplantation. 2006; 82: 1060-1066Crossref PubMed Scopus (121) Google Scholar; Kidd et al., 2010Kidd S. Caldwell L. Dietrich M. Samudio I. Spaeth E.L. Watson K. Shi Y. Abbruzzese J. Konopleva M. Andreeff M. Marini F.C. Mesenchymal stromal cells alone or expressing interferon-beta suppress pancreatic tumors in vivo, an effect countered by anti-inflammatory treatment.Cytotherapy. 2010; 12: 615-625Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar; Tian et al., 2010Tian K. Yang S. Ren Q. Han Z. Lu S. Ma F. Zhang L. Han Z. p38 MAPK contributes to the growth inhibition of leukemic tumor cells mediated by human umbilical cord mesenchymal stem cells.Cell. Physiol. Biochem. 2010; 26: 799-808Crossref PubMed Scopus (41) Google Scholar; Zhu et al., 2009Zhu Y. Sun Z. Han Q. Liao L. Wang J. Bian C. Li J. Yan X. Liu Y. Shao C. Zhao R.C. Human mesenchymal stem cells inhibit cancer cell proliferation by secreting DKK-1.Leukemia. 2009; 23: 925-933Crossref PubMed Scopus (256) Google Scholar), but others that the MSCs promoted tumor growth or metastases (Djouad et al., 2003Djouad F. Plence P. Bony C. Tropel P. Apparailly F. Sany J. Noël D. Jorgensen C. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals.Blood. 2003; 102: 3837-3844Crossref PubMed Scopus (1021) Google Scholar; Karnoub et al., 2007Karnoub A.E. Dash A.B. Vo A.P. Sullivan A. Brooks M.W. Bell G.W. Richardson A.L. Polyak K. Tubo R. Weinberg R.A. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis.Nature. 2007; 449: 557-563Crossref PubMed Scopus (2514) Google Scholar; Kurtova et al., 2009Kurtova A.V. Balakrishnan K. Chen R. Ding W. Schnabl S. Quiroga M.P. Sivina M. Wierda W.G. Estrov Z. Keating M.J. et al.Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance.Blood. 2009; 114: 4441-4450Crossref PubMed Scopus (257) Google Scholar; Patel et al., 2010Patel S.A. Meyer J.R. Greco S.J. Corcoran K.E. Bryan M. Rameshwar P. Mesenchymal stem cells protect breast cancer cells through regulatory T cells: role of mesenchymal stem cell-derived TGF-beta.J. Immunol. 2010; 184: 5885-5894Crossref PubMed Scopus (300) Google Scholar). Recently, we observed that incubation of human MSCs (hMSCs) with recombinant human tumor necrosis factor-α (TNF-α) activated the cells to express a number of potentially therapeutic proteins including tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) (Rahman et al., 2009Rahman M. Davis S.R. Pumphrey J.G. Bao J. Nau M.M. Meltzer P.S. Lipkowitz S. TRAIL induces apoptosis in triple-negative breast cancer cells with a mesenchymal phenotype.Breast Cancer Res. Treat. 2009; 113: 217-230Crossref PubMed Scopus (141) Google Scholar). TRAIL causes apoptosis in many malignant cells but not in normal cells; for this reason, soluble recombinant TRAIL was employed in a series of clinical trials (Gazitt, 1999Gazitt Y. TRAIL is a potent inducer of apoptosis in myeloma cells derived from multiple myeloma patients and is not cytotoxic to hematopoietic stem cells.Leukemia. 1999; 13: 1817-1824Crossref PubMed Scopus (129) Google Scholar; Johnstone et al., 2008Johnstone R.W. Frew A.J. Smyth M.J. The TRAIL apoptotic pathway in cancer onset, progression and therapy.Nat. Rev. Cancer. 2008; 8: 782-798Crossref PubMed Scopus (718) Google Scholar; Kelley et al., 2001Kelley S.K. Harris L.A. Xie D. Deforge L. Totpal K. Bussiere J. Fox J.A. Preclinical studies to predict the disposition of Apo2L/tumor necrosis factor-related apoptosis-inducing ligand in humans: characterization of in vivo efficacy, pharmacokinetics, and safety.J. Pharmacol. Exp. Ther. 2001; 299: 31-38PubMed Google Scholar), but the success was limited by the short half-life in serum (Kelley et al., 2001Kelley S.K. Harris L.A. Xie D. Deforge L. Totpal K. Bussiere J. Fox J.A. Preclinical studies to predict the disposition of Apo2L/tumor necrosis factor-related apoptosis-inducing ligand in humans: characterization of in vivo efficacy, pharmacokinetics, and safety.J. Pharmacol. Exp. Ther. 2001; 299: 31-38PubMed Google Scholar) and the lower bioactivity of the soluble protein compared to the membrane-bound form (Rus et al., 2005Rus V. Zernetkina V. Puliaev R. Cudrici C. Mathai S. Via C.S. Increased expression and release of functional tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by T cells from lupus patients with active disease.Clin. Immunol. 2005; 117: 48-56Crossref PubMed Scopus (28) Google Scholar). One strategy to overcome the limitations of soluble TRAIL is to use hMSCs as delivery vectors and thereby capitalize on their ability to home to tumors. hMSCs that were transduced with viral vectors to overexpress TRAIL suppressed tumor xenografts in several in vivo models including glioma, colorectal carcinoma, and metastatic breast cancer (Grisendi et al., 2010Grisendi G. Bussolari R. Cafarelli L. Petak I. Rasini V. Veronesi E. De Santis G. Spano C. Tagliazzucchi M. Barti-Juhasz H. et al.Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy.Cancer Res. 2010; 70: 3718-3729Crossref PubMed Scopus (215) Google Scholar; Loebinger et al., 2009Loebinger M.R. Eddaoudi A. Davies D. Janes S.M. Mesenchymal stem cell delivery of TRAIL can eliminate metastatic cancer.Cancer Res. 2009; 69: 4134-4142Crossref PubMed Scopus (350) Google Scholar; Menon et al., 2009Menon L.G. Kelly K. Yang H.W. Kim S.K. Black P.M. Carroll R.S. Human bone marrow-derived mesenchymal stromal cells expressing S-TRAIL as a cellular delivery vehicle for human glioma therapy.Stem Cells. 2009; 27: 2320-2330Crossref PubMed Scopus (162) Google Scholar; Mohr et al., 2008Mohr A. Lyons M. Deedigan L. Harte T. Shaw G. Howard L. Barry F. O'Brien T. Zwacka R. Mesenchymal stem cells expressing TRAIL lead to tumour growth inhibition in an experimental lung cancer model.J. Cell. Mol. Med. 2008; 12: 2628-2643Crossref PubMed Scopus (95) Google Scholar; Mueller et al., 2011Mueller L.P. Luetzkendorf J. Widder M. Nerger K. Caysa H. Mueller T. TRAIL-transduced multipotent mesenchymal stromal cells (TRAIL-MSC) overcome TRAIL resistance in selected CRC cell lines in vitro and in vivo.Cancer Gene Ther. 2011; 18: 229-239Crossref PubMed Scopus (71) Google Scholar). The use of viral vectors, however, introduces limitations such as insertional mutagenesis and phenotypic changes in the hMSCs. We also observed that DKK3 expression was increased upon exposure of hMSCs to TNF-α. DKK3 is suppressed in many breast cancer cell lines because the gene promoter is hypermethylated (Kuphal et al., 2006Kuphal S. Lodermeyer S. Bataille F. Schuierer M. Hoang B.H. Bosserhoff A.K. Expression of Dickkopf genes is strongly reduced in malignant melanoma.Oncogene. 2006; 25: 5027-5036Crossref PubMed Scopus (147) Google Scholar), an observation suggesting that DKK3 is a tumor suppressor gene. Furthermore, several reports showed that epigenetic inactivation of DKK3 stimulates the Wnt/β-catenin pathway that plays an important role in tumorigenesis (Bafico et al., 2004Bafico A. Liu G. Goldin L. Harris V. Aaronson S.A. An autocrine mechanism for constitutive Wnt pathway activation in human cancer cells.Cancer Cell. 2004; 6: 497-506Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar; Clevers, 2006Clevers H. Wnt/beta-catenin signaling in development and disease.Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4491) Google Scholar; Vogelstein and Kinzler, 2004Vogelstein B. Kinzler K.W. Cancer genes and the pathways they control.Nat. Med. 2004; 10: 789-799Crossref PubMed Scopus (3327) Google Scholar). This inactivation promotes the growth of human breast, lung, and cervical cancer (Lee et al., 2009aLee E.J. Jo M. Rho S.B. Park K. Yoo Y.N. Park J. Chae M. Zhang W. Lee J.H. Dkk3, downregulated in cervical cancer, functions as a negative regulator of beta-catenin.Int. J. Cancer. 2009; 124: 287-297Crossref PubMed Scopus (125) Google Scholar; Veeck et al., 2008Veeck J. Bektas N. Hartmann A. Kristiansen G. Heindrichs U. Knüchel R. Dahl E. Wnt signalling in human breast cancer: expression of the putative Wnt inhibitor Dickkopf-3 (DKK3) is frequently suppressed by promoter hypermethylation in mammary tumours.Breast Cancer Res. 2008; 10: R82Crossref PubMed Scopus (74) Google Scholar; Yue et al., 2008Yue W. Sun Q. Dacic S. Landreneau R.J. Siegfried J.M. Yu J. Zhang L. Downregulation of Dkk3 activates beta-catenin/TCF-4 signaling in lung cancer.Carcinogenesis. 2008; 29: 84-92Crossref PubMed Scopus (147) Google Scholar). Because hMSCs activated with TNF-α expressed both TRAIL and DKK3, we tested the hypothesis that activated hMSCs are tumor suppressive. Here, we show that preactivated hMSCs reduced the tumor burden in a lung metastatic xenograft model that was produced with MDA-MB-231 (MDA) in vivo. They also induced apoptosis of MDA cells and several other TRAIL-sensitive cancer cell lines and prevented cell-cycle progression of MDA cells in vitro. First, we observed that expression of TRAIL and DKK3 protein was upregulated in hMSCs after incubating the cells with 10 ng/ml TNF-α (Figure 1A and Figures S1A and 1B, available online). To explore whether hMSCs preactivated with TNF-α (preactivated hMSCs) have the ability to induce cell death in cancer cells, a xenograft model of human breast cancer metastasis with progressive tumor growth (Figure S1B) was induced by injecting MDA cells (2 × 106) intravenously into NOD/SCID mice (Figure 1C). The model was previously shown to respond to hMSCs virally transduced to express TRAIL (Loebinger et al., 2009Loebinger M.R. Eddaoudi A. Davies D. Janes S.M. Mesenchymal stem cell delivery of TRAIL can eliminate metastatic cancer.Cancer Res. 2009; 69: 4134-4142Crossref PubMed Scopus (350) Google Scholar). A week after MDA injection, either male control hMSCs (2 × 106) or male preactivated hMSCs (2 × 106) were intravenously injected weekly for 4 or 9 consecutive weeks. Both preactivated hMSCs and control hMSCs were found by immunofluorescence (IF) staining in the tumors 1 day after IV injection (Figure S1C). Some of the cells migrated to tumor sites and incorporated into the tumors (Figure S1C). However, the cells did not persist; after 1 week, less than 0.01% of the infused cells were detected by qPCR for the human Y chromosome in the injected male hMSCs (not shown). Therefore, we were able to employ qPCR for repetitive human Alu sequences to provide quantitative estimates of the growth of the human female breast cancer cells in the mouse lung. We did not examine the distribution of cancer cells and hMSCs in other organs. The results demonstrated that preactivated hMSCs suppressed tumor cell growth compared to HBSS control group at both early and late time points (Figure 1D). Controls of hMSCs that were not exposed to TNF-α and that did not express TRAIL (Figure 1A and S1A) did not have any statistically significant effect on tumor burden compared to the HBSS control group (Figure 1D). The gross pictures and histology images (H&E staining) of lungs demonstrated that injection of preactivated hMSCs decreased the number of tumor nodules (Figures 1E and 1F). The decrease in tumor burden seemed larger by gross images and histology of the lung (Figures 1E and 1F) than by the assays for Alu sequence (Figure 1D), perhaps because the assays for Alu sequences underestimated the tumor burden in the control samples as a result of necrosis and DNA degradation at the center of large tumors. Therefore, the results suggested that the hMSCs suppressed the tumors by homing to the tumor site. To determine whether hMSCs can induce apoptosis in MDA cells in vitro, MDA cells were directly cocultured with hMSCs, hMSCs in the presence of TNF-α (activated hMSCs), or preactivated hMSCs. After 24 hr coculture, hMSCs and MDA cells were distinguished by antibodies to CD90, an epitope expressed by hMSCs but not by MDA cells (Figure 2A ), and apoptosis of MDA cells was analyzed using 7AAD and Annexin V staining (Figure 2B). When MDA cells were cultured with activated hMSCs or preactivated hMSCs, the apoptosis increased remarkably with a corresponding decrease in the number of live MDA cells (Figure 2C). Naive hMSCs cocultured with MDA cells also reduced the number of live MDA cells, but to a lesser extent than activated hMSCs (Figure 2C). In addition, TRAIL expression in hMSCs was induced by incubation of the cells with two other proinflammatory agents, LPS and IFN-γ (Figures S2A and S2B). hMSCs preincubated with LPS or IFN-γ induced apoptosis of MDA cells in cocultures (Figures S2C and S2D). However, preactivated hMSCs with LPS induced more cell death because the upregulation of TRAIL by LPS was 500-fold whereas the upregulation by IFN-γ was only 30-fold. We also cocultured activated hMSCs with other TRAIL-sensitive cancer cell lines (Figure 2D). The activated hMSCs were effective in reducing the live cell number in two triple-negative breast cancer (TNBC) cell lines (HCC38 and MDA-MB-436) in addition to MDA, a pancreatic cancer cell line (CFPAC), a cervical cancer cell line (HeLa), and carcinomic human alveolar basal epithelial cells (A549). Activated hMSCs had no effect on a line of glioblastoma cells (U87) even though MSCs transduced to express TRAIL were previously shown to inhibit intracranial U87 glioma growth (Menon et al., 2009Menon L.G. Kelly K. Yang H.W. Kim S.K. Black P.M. Carroll R.S. Human bone marrow-derived mesenchymal stromal cells expressing S-TRAIL as a cellular delivery vehicle for human glioma therapy.Stem Cells. 2009; 27: 2320-2330Crossref PubMed Scopus (162) Google Scholar). The discrepancy is probably explained by the observation that U87 cells are less sensitive to rhTRAIL than MDA cells (Figure S2E). The results suggest that preconditioning hMSCs to express TRAIL can be useful, but gene modification may be necessary to obtain optimal therapeutic benefits in some circumstances. We elected to focus on the three triple-negative breast cancer cell lines. Induction of apoptosis by hMSCs in all three cell lines was partially reduced when TRAIL activity was inhibited by a TRAIL blocking antibody: MDA-MB-231 (denoted as MDA in Figures 2E and 2F), HCC38, and MDA-MB-436 cells (Figure S2F). The antibody was more effective in blocking the effects of rhTRAIL than in blocking the effects of preactivated hMSCs (Figures 2E and 2F), apparently because the hMSCs were continually activated to express TRAIL in the coculture system. Also, inhibition of a decoy receptor for TRAIL (osteoprotegerin; OPG) in hMSCs with troglitazone (Krause et al., 2010Krause U. Harris S. Green A. Ylostalo J. Zeitouni S. Lee N. Gregory C.A. Pharmaceutical modulation of canonical Wnt signaling in multipotent stromal cells for improved osteoinductive therapy.Proc. Natl. Acad. Sci. USA. 2010; 107: 4147-4152Crossref PubMed Scopus (93) Google Scholar) increased the apoptosis compared to control (Figure 2G). The results indicated that activated hMSCs induced TRAIL-dependent apoptosis in the three triple-negative breast cancer cell lines. The activation of hMSCs by TNF-α to induce apoptosis of MDA cells in coculture was concentration dependent over the range of 0.1 to 10 ng/ml (Figure 3A ). Activation of hMSCs with as little as 0.1 ng/ml TNF-α was adequate to induce MDA apoptosis. Cell-to-cell contact was required, since the hMSCs had no effect in transwell cocultures (Figure 3B) and an increase in soluble TRAIL was not detected in conditioned medium from the cells (Figure S3A), suggesting that TRAIL expressed by hMSCs was transmembrane. The apoptosis of MDA cells in cocultures increased with increasing ratios of hMSCs to MDA cells over the range of 0.06:1 to 2:1 (Figure 3C). Control experiments demonstrated that human foreskin fibroblasts (Hs68) did not express TRAIL upon incubation with TNF-α (Figure S3B) and they did not induce apoptosis of MDA cells upon coculture (Figure S3C). Two other samples of primary preparations of human dermal fibroblasts (hDF) slightly decreased the number of live MDA cells when cocultured with the MDA cells (Figures S3D and S3E) and TNF-α, but the effect was not inhibited by a blocking antibody to TRAIL (Figure S3F). As reported previously, there were variations in the quality of hMSCs obtained from bone marrow aspirates, even if the aspirates were drawn from the same normal volunteer at the same session and the hMSCs were isolated and expanded with a standardized protocol (Phinney et al., 1999Phinney D.G. Kopen G. Righter W. Webster S. Tremain N. Prockop D.J. Donor variation in the growth properties and osteogenic potential of human marrow stromal cells.J. Cell. Biochem. 1999; 75: 424-436Crossref PubMed Scopus (402) Google Scholar; Sekiya et al., 2002Sekiya I. Larson B.L. Smith J.R. Pochampally R. Cui J.G. Prockop D.J. Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality.Stem Cells. 2002; 20: 530-541Crossref PubMed Scopus (817) Google Scholar; Wolfe et al., 2008Wolfe M. Pochampally R. Swaney W. Reger R.L. Isolation and culture of bone marrow-derived human multipotent stromal cells (hMSCs).Methods Mol. Biol. 2008; 449: 3-25PubMed Google Scholar). Therefore, we compared four preparations of hMSCs, identified by their anonymous donor numbers. The four samples of preactivated hMSCs demonstrated large variations in the apoptosis induced in the MDA cells (Figure 3D). As expected, the apoptosis induced by the hMSCs correlated with their levels of TRAIL expression following incubation with TNF-α (Figure 3E). As observed previously, cultures of hMSCs lose many of their biological properties as they are expanded beyond about 20 population doublings in culture (Digirolamo et al., 1999Digirolamo C.M. Stokes D. Colter D. Phinney D.G. Class R. Prockop D.J. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate.Br. J. Haematol. 1999; 107: 275-281Crossref PubMed Scopus (750) Google Scholar; Larson et al., 2010Larson B.L. Ylostalo J. Lee R.H. Gregory C. Prockop D.J. Sox11 is expressed in early progenitor human multipotent stromal cells and decreases with extensive expansion of the cells.Tissue Eng. Part A. 2010; 16: 3385-3394Crossref PubMed Scopus (51) Google Scholar). As expected, hMSCs gradually lost their ability to express TRAIL upon TNF-α activation (Figure 3F) and to induce apoptosis of MDA cells as they were expanded through 20 or 25 population doublings (Figure 3G). These observations demonstrated that apoptosis induced by TNF-α activated hMSCs required upregulation of TRAIL and that the effectiveness of the cells varies with the quality of the hMSCs. To examine synergistic interactions between TRAIL-expressing activated hMSCs and chemotherapeutic drugs, we treated MDA cells with both doxorubicin and hMSCs or activated hMSCs. As reported previously (Mallory et al., 2005Mallory J.C. Crudden G. Oliva A. Saunders C. Stromberg A. Craven R.J. A novel group of genes regulates susceptibility to antineoplastic drugs in highly tumorigenic breast cancer cells.Mol. Pharmacol. 2005; 68: 1747-1756Crossref PubMed Scopus (43) Google Scholar), doxorubicin in a low concentration of 100 ng/ml (0.2 μM) suppressed proliferation of MDA as indicated by the decrease in recovery of live cells (Figure 3I) but did not induce apoptosis (Figure 3H). Incubation of MDA cells with doxorubicin decreased the number of live MDA cells recovered from cultures after 24 hr (Figure 3I) in a dose-dependent manner (Figure S3G). Addition of hMSCs, however, together with 100 ng/ml doxorubicin both further decreased the number of live MDA cells recovered from the cultures (Figure 3I) and greatly increased apoptosis (Figure 3H). The effect was synergistic in that the decrease in live MDA cells was greater than the additive effect observed with doxorubicin alone (Figure 3H) and activated hMSCs alone (Figure 2B). Of special note, the hMSCs were effective regardless of TNF-α activation (Figures 3H and 3I). Since doxorubicin enhances TRAIL-induced apoptosis by activating caspase or TRAIL receptors on cancer cells (Buchsbaum et al., 2003Buchsbaum D.J. Zhou T. Grizzle W.E. Oliver P.G. Hammond C.J. Zhang S. Carpenter M. LoBuglio A.F. Antitumor efficacy of TRA-8 anti-DR5 monoclonal antibody alone or in combination with chemotherapy and/or radiation therapy in a human breast cancer model.Clin. Cancer Res. 2003; 9: 3731-3741PubMed Google Scholar; Keane et al., 1999Keane M.M. Ettenberg S.A. Nau M.M. Russell E.K. Lipkowitz S. Chemotherapy augments TRAIL-induced apoptosis in breast cell lines.Cancer Res. 1999; 59: 734-741PubMed Google Scholar; Singh et al., 2003Singh T.R. Shankar S. Chen X. Asim M. Srivastava R.K. Synergistic interactions of chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand on apoptosis and on regression of breast carcinoma in vivo.Cancer Res. 2003; 63: 5390-5400PubMed Google Scholar), the low level of TRAIL activation in hMSCs, which was induced by the coculture with MDA even without TNF-α (Figure S3H), might be sufficient to induce the apoptosis in MDA cells, and then these dead cells create feedforward stimulation of TRAIL. This synergistic effect was replicated in two additional triple-negative breast cancer cell lines HCC38 and MDA-MB-436 (Figure S3I). Therefore, combination treatment of a chemotherapeutic drug and activated hMSCs can create synergistic effects and preactivation of hMSCs with proinflammatory cytokines may not be essential to induce apoptosis in MDA cells exposed to doxorubicin. Apoptosis of MDA cells by activated hMSCs appeared to increase with time in culture (Figure 4A ). Therefore, we assayed the levels of TRAIL in hMSCs isolated from the cocultures. There was a 10-fold increase in the expression of TRAIL in hMSCs recovered from cocultures of MDA cells and activated hMSCs (Figures 4B and 4C). The results suggested the hypothesis that apoptotic MDA cells might enhance expression of TRAIL in hMSCs. To test the hypothesis, we incubated hMSCs with apoptotic MDA cells. The apoptotic MDA cells were prepared by incubation with 100 ng/ml of recombinant human TRAIL (rhTRAIL) for 24 hr in serum-free media (Figure S4A) and recovery of nonadherent cells from the cultures. As expected, apoptotic MDA cells enhanced TRAIL expression in TNF-α activated hMSCs to the same extent as in the coculture system (compare Figure 4D to 4B). We then tested the hypothesis that the effects of the apoptotic MDA cells were explained by RNA that is released from damaged tissue (Karikó et al., 2004Karikó K. Ni H. Capodici J. Lamphier M. Weissman D. mRNA is an endogenous ligand for Toll-like receptor 3.J. Biol. Chem. 2004; 279: 12542-12550Crossref PubMed Scopus (825) Google Scholar). We assayed hMSCs for expression of TLR3, a specific receptor for RNA (Karikó et al., 2004Karikó K. Ni H. Capodici J. Lamphier M. Weissman D. mRNA is an endogenous ligand for Toll-like receptor 3.J. Biol. Chem. 2004; 279: 12542-12550Crossref PubMed Scopus (825) Google Scholar) that increases NF-κB signaling and thereby triggers an essential step in the pathway for induction of TRAIL (Rivera-Walsh et al., 2001Rivera-Walsh I. Waterfield M. Xiao G. Fong A. Sun S.C. NF-kappaB signaling pathway governs TRAIL gene expression and human T-cell leukemia virus-I Tax-induced T-cell death.J. Biol. Chem. 2001; 276: 40385-40388Crossref PubMed Scopus (84) Google Scholar). Expression of TLR3 in hMSCs was increased by incubation with TNF-α and further enhanced by coculture of the activated hMSCs with MDA cells (Figures 4E). Increased expression of TLR3 was also observed when hMSCs were treated with apoptotic MDA cells (Figure 4F). Treatment of apoptotic MDA cells with RNase inhibited the increase of TRAIL in hMSCs (Figure 4G). Treatment with DNase also inhibited the increase of TRAIL in hMSCs; however, the expression level of TLR9, a receptor for DNA (Zhang et al., 2010Zhang Q. Raoof M. Chen Y. Sumi Y. Sursal T. Junger W. Brohi K. Itagaki K. Hauser C.J. Circulating mitochondrial DAMPs cause inflammatory responses to injury.Nature. 2010; 464: 104-107Crossref PubMed Scopus (2478) Google Scholar), was low in hMSCs and was not upregulated by treatment of TNF-α or apoptotic MDA cells (data not shown). The roles of RNA and TLR3 were confirmed by the observations that poly(I:C), a synthetic ligand for TLR3 (Alexopoulou et al., 2001Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4909) Google Scholar), increased expression of TRAIL in hMSCs (Figures 4H and S4B) and caused a small but statistically significant increase in MDA apoptosis when added to cocultures (Figure 4I). Furthermore, adding a TLR3 blocking antibody reduced apoptosis of MDA cells in the coculture system and led to recovery of greater numbers of live MDA cells (Figure 4J and S4C). The results suggested that the further increase of TRAIL in hMSCs observed in cocultures with MDA cells was mediated by feedforward stimulation of TLR3 by RNA, by DNA, and probably by other DAMPs from apoptotic MDA cells. In the coculture system, preactivated hMSCs also inhibited cell-cycle progression in the recovered adherent viable MDA cells (Figures 5A and S5A). In transwell cocultures, the inhibition was less: 3.3% increase in G1 (Figures S5B and S5C) versus 17.4% in cocultures with direct contact between the cells (Figures 5A and S5A). The results therefore suggested t" @default.
• W2160392015 created "2016-06-24" @default.
• W2160392015 creator A5030828941 @default.
• W2160392015 creator A5063133761 @default.
• W2160392015 creator A5071417704 @default.
• W2160392015 creator A5079754427 @default.
• W2160392015 date "2012-12-01" @default.
• W2160392015 modified "2023-10-14" @default.
• W2160392015 title "Preactivation of Human MSCs with TNF-α Enhances Tumor-Suppressive Activity" @default.
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