Mesenchymal stem cell-based tumor-targeted gene therapy in gastrointestinal cancer.

Mesenchymal stem (or stromal) cells (MSCs) are nonhematopoietic progenitor cells that can be obtained from bone marrow aspirates or adipose tissue, expanded and genetically modified in vitro, and then used for cancer therapeutic strategies in vivo. Here, we review available data regarding the application of MSC-based tumor-targeted therapy in gastrointestinal cancer, provide an overview of the general history of MSC-based gene therapy in cancer research, and discuss potential problems associated with the utility of MSC-based therapy such as biosafety, immunoprivilege, transfection methods, and distribution in the host.

Related papers:

title: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.
abstract: The considerable therapeutic potential of human multipotent mesenchymal stromal cells (MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However, investigators report studies of MSC using different methods of isolation and expansion, and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes, which hinders progress in the field. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First, MSC must be plastic-adherent when maintained in standard culture conditions. Second, MSC must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. Third, MSC must differentiate to osteoblasts, adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds, we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators.

title: Multilineage potential of adult human mesenchymal stem cells.
abstract: Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.

title: Improved Arteriogenesis with Simultaneous Skeletal Muscle Repair in Ischemic Tissue by SCL+ Multipotent Adult Progenitor Cell Clones from Peripheral Blood
abstract: The CD34– murine stem cell line RM26 cloned from peripheral blood mononuclear cells has been shown to generate hematopoietic progeny in lethally irradiated animals. The peripheral blood-derived cell clones expresses a variety of mesodermal and erythroid/myeloid transcription factors suggesting a multipotent differentiation potential like the bone marrow-derived ‘multipotent adult progenitor cells’ (MAP-C).  SCL+ CD34– RM26 cells were transfused intravenously into mice suffering from chronic hind-limb ischemia, evaluating the effect of stem cells on collateral artery growth and simultaneous skeletal muscle repair.  RM26 cells are capable of differentiating in vitro into endothelial cells when cultured on the appropriate collagen matrix. Activation of the SCL stem cell enhancer (SCL+) is mediated through the binding to two Ets and one GATA site and cells start to express milieu- and growth condition-dependent levels of the endothelial markers CD31 (PECAM) and Flt-1 (VEGF-R1). Intravenously infused RM26 cells significantly improved the collateral blood flow (arteriogenesis) and neo-angiogenesis formation in a murine hind-limb ischemia transplant model. Although transplanted RM26 cells did not integrate into the growing collateral arteries, cells were found adjacent to local arteriogenesis, but instead integrated into the ischemic skeletal muscle exclusively in the affected limb for simultaneous tissue repair.  These data suggest that molecularly primed hem-/mesangioblast-type adult progenitor cells can circulate in the peripheral blood improving perfusion of tissues with chronic ischemia and extending beyond the vascular compartment.

title: Mesenchymal Stem Cells Differentiate into an Endothelial Phenotype, Enhance Vascular Density, and Improve Heart Function in a Canine Chronic Ischemia Model
abstract: Bone marrow–derived stem cells are under investigation as a treatment for ischemic heart disease. Mesenchymal stem cells (MSCs) have been used preferentially in the acute ischemia model; data in the chronic ischemia model are lacking. Twelve dogs underwent ameroid constrictor placement. Thirty days later, they received intramyocardial injections of either MSCs (100×106 MSCs/10 mL saline) (n=6) or saline only (10 mL) (controls) (n=6). All were euthanized at 60 days. Resting and stress 2D echocardiography was performed at 30 and 60 days after ameroid placement. White blood cell count (WBC), C-reactive protein (CRP), creatine kinase MB (CK-MB), and troponin I levels were measured. Histopathological and immunohistochemical analyses were performed. Mean left ventricular ejection fraction was similar in both groups at baseline but significantly higher in treated dogs at 60 days. WBC and CRP levels were similar over time in both groups. CK-MB and troponin I increased from baseline to 48 hours, eventually returning to baseline. There was a trend toward reduced fibrosis and greater vascular density in the treated group. MSCs colocalized with endothelial and smooth muscle cells but not with myocytes. In a canine chronic ischemia model, MSCs differentiated into smooth muscle cells and endothelial cells, resulting in increased vascularity and improved cardiac function.

title: Genetic engineering of mesenchymal stem cells and its application in human disease therapy.
abstract: The use of stem cells for tissue regeneration and repair is advancing both at the bench and bedside. Stem cells isolated from bone marrow are currently being tested for their therapeutic potential in a variety of clinical conditions including cardiovascular injury, kidney failure, cancer, and neurological and bone disorders. Despite the advantages, stem cell therapy is still limited by low survival, engraftment, and homing to damage area as well as inefficiencies in differentiating into fully functional tissues. Genetic engineering of mesenchymal stem cells is being explored as a means to circumvent some of these problems. This review presents the current understanding of the use of genetically engineered mesenchymal stem cells in human disease therapy with emphasis on genetic modifications aimed to improve survival, homing, angiogenesis, and heart function after myocardial infarction. Advancements in other disease areas are also discussed.

title: The therapeutic potential of mesenchymal stem cells
abstract: Mesenchymal stem cells (MSCs) are multipotent cells with a number of potential therapeutic applications. At present, they are being used in a clinical trial for the treatment of myocardial infarction and are being studied as a therapy for other vascular disorders. Treatments of neurologic disorders and anticancer therapy with MSCs have progressed in light of the migratory properties of MSCs to brain injury and tumors. The osteogenic potential of MSCs is being exploited in work investigating their use in bone regeneration therapy, and the immunomodulatory function of MSCs is being evaluated as a possible therapy for graft-versus-host disease. Here, the authors review recent work contributing to the knowledge of MSC biology and the advances in gene therapy and tissue regeneration using MSCs.

title: Therapeutic potential of genetically modified mesenchymal stem cells
abstract: Bone marrow-derived mesenchymal stem cells (MSC) are multipotent adult stem cells of mesodermal origin localized within the bone marrow compartment. MSC possess multilineage property making them useful for a number of potential therapeutic applications. MSC can be isolated from the bone marrow, expanded in culture and genetically modified to serve as cell carriers for local or systemic therapy. Despite their ability to differentiate into osteoblasts, chondrocytes, adipocytes, myocytes and neuronal cells under appropriate stimuli, distinct molecular signals that guide migration of MSC to specific targets largely remain unknown. The pluripotent nature of MSC makes them ideal resources for regenerative medicine, graft-versus-host disease and autoimmune diseases. Despite their therapeutic potential in a variety of diseases, certain issues need to be critically addressed both in in vitro expansion of these cells without losing their stem cell properties, and the long-term fate of the transplanted MSC in vivo following ex vivo modifications. Finally, understanding of complex, multistep and multifactorial differentiation pathways from pluripotent stem cells to functional tissues will allow us to manipulate MSC for the formation of competent composite tissues in situ. The present article will provide comprehensive account of the characteristics of MSC, their isolation and culturing, multilineage properties and potential therapeutic applications.

title: Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep
abstract: Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.

title: Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta
abstract: In principle, transplantation of mesenchymal progenitor cells would attenuate or possibly correct genetic disorders of bone, cartilage and muscle, but clinical support for this concept is lacking. Here we describe the initial results of allogeneic bone marrow transplantation in three children with osteogenesis imperfecta, a genetic disorder in which osteoblasts produce defective type I collagen, leading to osteopenia, multiple fractures, severe bony deformities and considerably shortened stature. Three months after osteoblast engraftment (1.5–2.0% donor cells), representative specimens of trabecular bone showed histologic changes indicative of new dense bone formation. All patients had increases in total body bone mineral content ranging from 21 to 29 grams (median, 28), compared with predicted values of 0 to 4 grams (median, 0) for healthy children with similar changes in weight. These improvements were associated with increases in growth velocity and reduced frequencies of bone fracture. Thus, allogeneic bone marrow transplantation can lead to engraftment of functional mesenchymal progenitor cells, indicating the feasibility of this strategy in the treatment of osteogenesis imperfecta and perhaps other mesenchymal stem cell disorders as well.

title: Adipose tissue-derived human mesenchymal stem cells mediated prodrug cancer gene therapy.
abstract: Human adipose tissue-derived mesenchymal stem cells (AT-MSC) are considered to be a promising source of autologous stem cells in personalized cell-based therapies. Tumor tracking properties of MSC provide an attractive opportunity for targeted transgene delivery into the sites of tumor formation. In the present study, we addressed whether the suicide gene introduction into human AT-MSC could produce a tumor-specific prodrug converting cellular vehicle for targeted chemotherapy. We prepared yeast fusion cytosine deaminase::uracil phosphoribosyltransferase gene-expressing cells [cytosine deaminase (CD)-expressing AT-MSC (CD-AT-MSC)] by retrovirus transduction. We explored their therapeutic potential on a model of human colon cancer in the presence of prodrug 5-fluorocytosine (5-FC). Gene manipulation of human AT-MSC did not sensitize CD-AT-MSC to 5-FC, thus overcoming the inherent disadvantage of suicide effect on cellular vehicle. CD-AT-MSC in combination with 5-FC augmented the bystander effect and selective cytotoxicity on target tumor cells HT-29 in direct coculture in vitro. We confirmed directed migration ability of AT-MSC and CD-AT-MSC toward tumor cells HT-29 in vitro. Moreover, we achieved significant inhibition of s.c. tumor xenograft growth by s.c. or i.v. administered CD-AT-MSC in immunocompromised mice treated with 5-FC. We confirmed the ability of CD-AT-MSC to deliver the CD transgene to the site of tumor formation and mediate strong antitumor effect in vivo. Taken together, these data characterize MSC derived from adipose tissue as suitable delivery vehicles for prodrug converting gene and show their utility for a personalized cell-based targeted cancer gene therapy.

title: The (in) auspicious role of mesenchymal stromal cells in cancer: be it friend or foe.
abstract: Recent progress in the research of mesenchymal stromal cells/multipotent stromal cells (MSC) has revealed numerous beneficial innate characteristics, suggesting potential value in an array of cellular therapies. MSC are easily isolated from bone marrow (BM), fat and other tissues, and are readily propagated in vitro. Transplanted/injected MSC have been shown to migrate to a variety of organs and tissues; however, sites of inflammation and pathology elicit enhanced MSC homing for tissue remodeling and repair. Tumors utilize many of the same inflammatory mediators uncovered in wound healing and likewise provide a site for preferential MSC homing. Although incorporation into the tumor microenvironment is apparent, the role of recruited MSC in the tumor microenvironment remains unclear. Some published studies have shown enhancement of tumor growth and development, perhaps through immunomodulatory and pro-angiogenic properties, while others have shown no apparent effect or have demonstrated inhibition of tumor growth and extended survival. This controversy remains at the forefront as clinical applications of MSC commence in anti-tumor therapies as well as as adjuncts to stem cell transplantation and in ameliorating graft-versus-host disease. Careful analysis of past studies and thoughtful design of future experiments will help to resolve the discrepancies in the field and lead to clinical utility of MSC in disease treatment. This review highlights the current theories of the role of MSC in tumors and explores current controversies.

title: Inflammation and cancer
abstract: Recent data have expanded the concept that inflammation is a critical component of tumour progression. Many cancers arise from sites of infection, chronic irritation and inflammation. It is now becoming clear that the tumour microenvironment, which is largely orchestrated by inflammatory cells, is an indispensable participant in the neoplastic process, fostering proliferation, survival and migration. In addition, tumour cells have co-opted some of the signalling molecules of the innate immune system, such as selectins, chemokines and their receptors for invasion, migration and metastasis. These insights are fostering new anti-inflammatory therapeutic approaches to cancer development.

title: Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide.
abstract: Mesenchymal stem cells (MSCs) can become potently immunosuppressive through unknown mechanisms. We found that the immunosuppressive function of MSCs is elicited by IFNgamma and the concomitant presence of any of three other proinflammatory cytokines, TNFalpha, IL-1alpha, or IL-1beta. These cytokine combinations provoke the expression of high levels of several chemokines and inducible nitric oxide synthase (iNOS) by MSCs. Chemokines drive T cell migration into proximity with MSCs, where T cell responsiveness is suppressed by nitric oxide (NO). This cytokine-induced immunosuppression was absent in MSCs derived from iNOS(-/-) or IFNgammaR1(-/-) mice. Blockade of chemokine receptors also abolished the immunosuppression. Administration of wild-type MSCs, but not IFNgammaR1(-/-) or iNOS(-/-) MSCs, prevented graft-versus-host disease in mice, an effect reversed by anti-IFNgamma or iNOS inhibitors. Wild-type MSCs also inhibited delayed-type hypersensitivity, while iNOS(-/-) MSCs aggravated it. Therefore, proinflammatory cytokines are required to induce immunosuppression by MSCs through the concerted action of chemokines and NO.

title: The immunosuppressive effects of human bone marrow-derived mesenchymal stem cells target T cell proliferation but not its effector function.
abstract: Mesenchymal stem cells (MSC) are non-haematopoietic stem cells that are capable of differentiating into tissues of mesodermal origin. MSC play an important role in supporting the development of fetal and adult haematopoiesis. More recently, MSC have also been found to exhibit inhibitory effect on T cell responses. However, there is little information on the mechanism of this immunosuppression and our study addresses this issue by targeting T cell functions at various level of immune responses. We have generated MSC from human adult bone marrow (BM) and investigated their immunoregulatory function at different phases of T cell responses. MSC showed the ability to inhibit mitogen (CD3/CD28 microbeads)-activated T cell proliferation in a dose-dependent manner. In order to evaluate the specificity of this immunosuppression, the proliferation of CD4(+) and CD8(+) cells were measured. MSC equally inhibit CD4(+) and CD8(+) subpopulations of T cells in response to PHA stimulation. However, the antiproliferative effect of MSC is not due to the inhibition of T cell activation. The expression of early activation markers of T cells, namely CD25 and CD69 were not significantly altered by MSC at 24, 48 and 72h. Furthermore, the immunosuppressive effect of MSC mainly targets T cell proliferation rather than their effector function since cytotoxicity of T cells is not affected. This work demonstrates that the immunosuppressive effect of MSC is exclusively a consequence of an anti-proliferative activity, which targets T cells of different subpopulations. For this reason, they have the potential to be exploited in the control of unwanted immune responses such as graft versus host disease (GVHD) and autoimmunity.

title: Mesenchymal stem cells induce apoptosis of activated T cells
abstract: Mesenchymal stem cells (MSC) have recently been used successfully in humans to control severe graft-versus-host disease. However, the mechanisms involved in their immunomodulatory effects remain a matter of debate. Here, we show that MSC are unable to activate allogeneic T cells even in the presence of T-cell growth factors. We then found that MSC inhibit T-cell proliferation triggered either by allogeneic, mitogenic or antigen-specific stimuli. Interestingly, MSC inhibit T-cell proliferation by inducing apoptosis of activated T cells, but have no effect on resting T cells. Furthermore, we show that this apoptosis could be related to the conversion of tryptophan into kynurenine by indoleamine 2,3-dioxygenase expressed by MSC in the presence of IFNγ. Moreover, we show that the inhibitory effect of MSC is neither abrogated nor modified during expansion in culture or after irradiation. Together, these results bring new insight to the mechanisms of immunosuppression induced by MSC and might help to develop their clinical use controlling immune-related adverse effects in humans.

title: Mesenchymal Stem Cells Alter Migratory Property of T and Dendritic Cells to Delay the Development of Murine Lethal Acute Graft‐Versus‐Host Disease
abstract: Due to the potent immunoregulatory capacity, mesenchymal stem cells (MSCs) have been used in clinical trials to treat acute graft‐versus‐host disease (aGvHD), although the detailed in vivo mechanisms remain elusive. In a murine lethal aGvHD model, MSCs delayed the development of the disease. Interestingly, we found that MSC infusion increased the number of T lymphocytes in the secondary lymphoid organs (SLOs). Since the expression of CD62L and CCR7 is prerequisite for lymphocyte migration into SLOs, the in vitro experiments revealed that in the presence of MSCs, T lymphocytes (including CD4+CD25+ regulatory T cells) preferred to take the naive‐like phenotype (CD62L+/CCR7+) in mixed lymphocyte reaction and maintained the migratory activity elicited by secondary lymphoid tissue chemokine (SLC). Dendritic cells (DCs) are the initiator of immune response. CCR7 expression is pivotal for their maturation and migration into SLOs. However, CCR7 expression and SLC‐driven migratory activity of DCs were remarkably suppressed by MSC coculture. The processes above were realized mainly through secretory mechanism. Consistently, MSC infusion maintained T lymphocytes to take CD62L+/CCR7+ phenotype and decreased the CCR7 expression and proportion of DCs in SLOs of aGvHD mice. In conclusion, the altered migratory properties of T cells and DCs might contribute to the immunosuppressive activity of transplanted MSCs in the setting of aGvHD.

title: Embryonic endothelial progenitor cells armed with a suicide gene target hypoxic lung metastases after intravenous delivery.
abstract: We show that mouse embryonic endothelial progenitor cells (eEPCs) home preferentially to hypoxic lung metastases when administered intravenously. This specificity is inversely related to the degree of perfusion and vascular density in the metastasis and directly related to local levels of hypoxia and VEGF. Ex vivo expanded eEPCs that were genetically modified with a suicide gene specifically and efficiently eradicated lung metastases with scant patent blood vessels. eEPCs do not express MHC I proteins, are resistant to natural killer cell-mediated cytolysis, and can contribute to tumor vessel formation also in nonsyngeneic mice. These results indicate that eEPCs can be used in an allogeneic setting to treat hypoxic metastases that are known to be resistant to conventional therapeutic regimes.

title: Safety and antitumor activity of recombinant soluble Apo2 ligand.
abstract: TNF and Fas ligand induce apoptosis in tumor cells; however, their severe toxicity toward normal tissues hampers their application to cancer therapy. Apo2 ligand (Apo2L, or TRAIL) is a related molecule that triggers tumor cell apoptosis. Apo2L mRNA is expressed in many tissues, suggesting that the ligand may be nontoxic to normal cells. To investigate Apo2L's therapeutic potential, we generated in bacteria a potently active soluble version of the native human protein. Several normal cell types were resistant in vitro to apoptosis induction by Apo2L. Repeated intravenous injections of Apo2L in nonhuman primates did not cause detectable toxicity to tissues and organs examined. Apo2L exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon, lung, breast, kidney, brain, and skin cancer. Treatment of athymic mice with Apo2L shortly after tumor xenograft injection markedly reduced tumor incidence. Apo2L treatment of mice bearing solid tumors induced tumor cell apoptosis, suppressed tumor progression, and improved survival. Apo2L cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil or CPT-11, causing substantial tumor regression or complete tumor ablation. Thus, Apo2L may have potent anticancer activity without significant toxicity toward normal tissues.

title: Chemoprevention of Cancer
abstract: Cancer chemoprevention is defined as the use of natural, synthetic, or biologic chemical agents to reverse, suppress, or prevent carcinogenic progression to invasive cancer. The success of several recent clinical trials in preventing cancer in high‐risk populations suggests that chemoprevention is a rational and appealing strategy. This review will highlight current clinical research in chemoprevention, the biologic effects of chemopreventive agents on epithelial carcinogenesis, and the usefulness of intermediate biomarkers as markers of premalignancy. Selected chemoprevention trials are discussed with a focus on strategies of trial design and clinical outcome. Future directions in the field of chemoprevention will be proposed that are based on recently acquired mechanistic insight into carcinogenesis.

title: Why do so many cancer patients fail to respond to interferon therapy?
abstract: Since their first use in the clinic some 25 years ago, interferons (IFNs) have become accepted therapy in a range of cancer forms. However, although in some patients they induce remission, in the great majority they are of no benefit or, at best, lead only to minor improvements. This review considers possible reasons for these failures.

title: Interferon levels in human pulmonary tumors are lower than plasma levels.
abstract: It is uncertain whether interferon levels in the interstitial fluid of tumors are equivalent to interferon plasma levels and we have investigated this problem in human pulmonary tumors by infusing human recombinant interferon alpha A and natural interferon Beta for about three hours before surgery. By determining the hematocrit and hemoglobin content it was possible to calculate interferon values (International Units/g wet tissue) present in the interstitial fluid of tumor and lung samples, simultaneously. In 14 patients (epidermoids, n = 9 and adenocarcinomas, n = 5) interferon levels in tumor and "normal" lung expressed as percentages of interferon plasma levels were: 9.5 +/- 3.9 and 29.8 +/- 6.9 for recombinant interferon alpha A and 3.1 +/- 0.4 and 10.1 +/- 2.4 for natural interferon Beta, respectively. Differences for both interferons are statistically significant (p less than 0.05). To our knowledge these are the first data indicating that interferon levels in pulmonary tumor interstitial fluid are markedly lower than those in normal lung although they do not clarify the main factor responsible for the decrease, they explain at least in part the negligible therapeutic activity of interferons in these tumors and emphasize the need for new approaches for improving the therapeutic index of interferons.

title: A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme.
abstract: Previous uncontrolled clinical trials have shown the in vivo retrovirus (RV)-mediated transduction of glioblastoma cells with the herpes simplex virus thymidine kinase (HSV-tk) gene and subsequent systemic treatment with ganciclovir to be feasible and well tolerated. However, because of continued tumor progression in most patients, the antitumor effect could not be determined using historical controls. Here, we describe a phase III, multicenter, randomized, open-label, parallel-group, controlled trial of the technique in the treatment of 248 patients with newly diagnosed, previously untreated glioblastoma multiforme (GBM). Patients received, in equal numbers, either standard therapy (surgical resection and radiotherapy) or standard therapy plus adjuvant gene therapy during surgery. Progression-free median survival in the gene therapy group was 180 days compared with 183 days in control subjects. Median survival was 365 versus 354 days, and 12-month survival rates were 50 versus 55% in the gene therapy and control groups, respectively. These differences were not significant. Therefore, the adjuvant treatment improved neither time to tumor progression nor overall survival time, although the feasibility and good biosafety profile of this gene therapy strategy were further supported. The failure of this specific protocol may be due mainly to the presumably poor rate of delivery of the HSV-tk gene to tumor cells. In addition, the current mode of manual injection of vector-producing cells with a nonmigratory fibroblast phenotype limits the distribution of these cells and the released replication-deficient RV vectors to the immediate vicinity of the needle track. Further evaluation of the RV-mediated gene therapy strategy must incorporate refinements such as improved delivery of vectors and transgenes to the tumor cells, noninvasive in vivo assessment of transduction rates, and improved delivery of the prodrug across the blood-brain and blood-tumor barrier to the transduced tumor cells.

title: Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents.
abstract: High concentrations of interferon beta (IFN-beta) inhibit malignant cell growth in vitro. However, the therapeutic utility of IFN-beta in vivo is limited by its excessive toxicity when administered systemically at high doses. Mesenchymal stem cells (MSC) can be used to target delivery of agents to tumor cells. We tested whether MSC can deliver IFN-beta to tumors, reducing toxicity.



Human MSC were transduced with an adenoviral expression vector carrying the human IFN-beta gene (MSC-IFN-beta cells). Flow cytometry was used to measure tumor cell proliferation among in vitro co-cultures of MSC-IFN-beta cells and human MDA 231 breast carcinoma cells or A375SM melanoma cells. We used a severe combined immunodeficiency mouse xenograft model (4-10 mice per group) to examine the effects of injected MSC-IFN-beta cells and human recombinant IFN-beta on the growth of MDA 231- and A375SM-derived pulmonary metastases in vivo and on survival. All statistical tests were two-sided.



Co-culture of MSC-IFN-beta cells with A375SM cells or MDA 231 cells inhibited tumor cell growth as compared with growth of the tumor cells cultured alone (differences in mean percentage of control cell growth: -94.0% [95% confidence interval [CI] = -81.2% to -106.8%; P<.001] and -104.8% [95% CI = -82.1% to -127.5%; P<.001], respectively). Intravenous injection of MSC-IFN-beta cells into mice with established MDA 231 or A375SM pulmonary metastases led to incorporation of MSC in the tumor architecture and, compared with untreated control mice, to prolonged mouse survival (median survival for MDA 231-injected mice: 60 and 37 days for MSC-injected and control mice, respectively [difference = 23.0 days (95% CI = 14.5 to 34.0 days; P<.001]; median survival for A375SM-injected mice: 73.5 and 30.0 days for MSC-injected and control mice, respectively [difference = 43.5 days (95% CI = 37.0 to 57.5 days; P<.001]). By contrast, intravenous injection of recombinant IFN-beta did not prolong survival in the same models (median survival for MDA 231-injected mice: 41.0 and 37.0 days for IFN-beta-injected and control mice, respectively [difference = 4 days, 95% CI = -5 to 10 days; P = .308]; median survival for A375SM-injected mice: 32.0 and 30.0 days for IFN-beta-injected and control mice, respectively [difference = 2 days, 95% CI = 0 to 4.5 days; P = .059]).



Injected MSC-IFN-beta cells suppressed the growth of pulmonary metastases, presumably through the local production of IFN-beta in the tumor microenvironment. MSC may be an effective platform for the targeted delivery of therapeutic proteins to cancer sites.

title: Mesenchymal stem cell delivery of TRAIL can eliminate metastatic cancer.
abstract: Cancer is a leading cause of mortality throughout the world and new treatments are urgently needed. Recent studies suggest that bone marrow-derived mesenchymal stem cells (MSC) home to and incorporate within tumor tissue. We hypothesized that MSCs engineered to produce and deliver tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a transmembrane protein that causes selective apoptosis of tumor cells, would home to and kill cancer cells in a lung metastatic cancer model. Human MSCs were transduced with TRAIL and the IRES-eGFP reporter gene under the control of a tetracycline promoter using a lentiviral vector. Transduced and activated MSCs caused lung (A549), breast (MDAMB231), squamous (H357), and cervical (Hela) cancer cell apoptosis and death in coculture experiments. Subcutaneous xenograft experiments confirmed that directly delivered TRAIL-expressing MSCs were able to significantly reduce tumor growth [0.12 cm(3) (0.04-0.21) versus 0.66 cm(3) (0.21-1.11); P < 0.001]. We then found, using a pulmonary metastasis model, systemically delivered MSCs localized to lung metastases and the controlled local delivery of TRAIL completely cleared the metastatic disease in 38% of mice compared with 0% of controls (P < 0.05). This is the first study to show a significant reduction in metastatic tumor burden with frequent eradication of metastases using inducible TRAIL-expressing MSCs. This has a wide potential therapeutic role, which includes the treatment of both primary tumors and their metastases, possibly as an adjuvant therapy in clearing micrometastatic disease following primary tumor resection.

title: Human mesenchymal stem cells lack tumor tropism but enhance the antitumor activity of oncolytic adenoviruses in orthotopic lung and breast tumors.
abstract: Systemic adenoviral delivery into tumors is inefficient because of liver sequestration of intravenously administered virus. One potential solution for improving bioavailability is the use of carrier cells such as human mesenchymal stem cells (MSCs), which have been suggested to have inherent tumor tropism. Here we investigated the capacity of capsid-modified adenoviruses to infect and replicate in MSCs. Further, biodistribution and tumor-killing efficacy of MSCs loaded with oncolytic adenoviruses were evaluated in orthotopic murine models of lung and breast cancer. In vitro, heparan sulfate proteoglycan- and alpha(v)beta integrin-targeted viruses enhanced gene delivery to bone marrow- and adipose tissue-derived MSCs up to 11,000-fold over adenovirus serotype 5 (Ad5). Infectivity-enhanced oncolytic adenoviruses showed notably higher rates of cytolysis of in vitro-passaged MSCs in comparison with wild-type virus. In vivo, intravenously injected MSCs homed primarily to the lungs, and virus was released into advanced orthotopic breast and lung tumors for therapeutic efficacy and increased survival. When the same dose of virus was injected intravenously without MSCs, only transduction of the liver was seen. These results suggest that MSCs loaded with oncolytic adenoviruses might be a useful approach for improving the bioavailability of systemically administered oncolytic adenoviruses.

title: Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy.
abstract: Adipose-derived mesenchymal stromal/stem cells (AD-MSC) may offer efficient tools for cell-based gene therapy approaches. In this study, we evaluated whether AD-MSC could deliver proapoptotic molecules for cancer treatment. Human AD-MSCs were isolated and transduced with a retroviral vector encoding full-length human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a proapoptotic ligand that induces apoptosis in a variety of human cancers but not normal tissues. Although several studies have documented the antitumor activity of recombinant human TRAIL, its use in vivo is limited by a short half-life in plasma due to a rapid clearance by the kidney. We found that these limitations can be overcome using stably transduced AD-MSC, which could serve as a constant source of TRAIL production. AD-MSC armed with TRAIL targeted a variety of tumor cell lines in vitro, including human cervical carcinoma, pancreatic cancer, colon cancer, and, in combination with bortezomib, TRAIL-resistant breast cancer cells. Killing activity was associated with activation of caspase-8 as expected. When injected i.v. or s.c. into mice, AD-MSC armed with TRAIL localized into tumors and mediated apoptosis without significant apparent toxicities to normal tissues. Collectively, our results provide preclinical support for a model of TRAIL-based cancer therapy relying on the use of adipose-derived mesenchymal progenitors as cellular vectors.

title: Pigment epithelial-derived factor and melanoma differentiation associated gene-7 cytokine gene therapies delivered by adipose-derived stromal/mesenchymal stem cells are effective in reducing prostate cancer cell growth.
abstract: Adipose-derived stromal/mesenchymal stem cells (ASC) have gained interest as promising tools for delivering cancer therapy. Adipose tissue can be obtained readily in amounts sufficient for ASC isolation, which can be expanded rapidly, allowing its use at low passage numbers, and can be transduced by viral and nonviral means. Our goal was to examine the potential of ASC to deliver cytokine gene therapies melanoma differentiation associated gene-7 (MDA-7) or pigment epithelial-derived factor (PEDF) to cancer cells. These novel cytokines are a potent proapoptotic and an antiangiogenesis mediator, respectively, with potential as antitumor agents. Expression of cytokine therapies did not adversely affect ASC biology, and these cells were still able to differentiate and retain normal viability. The ASC cytokine therapies were efficient in reducing tumor cell growth in coculture and also in suppressing in vitro angiogenesis phenotypes. We also observed that ASC retained their innate ability to migrate toward tumor cells in coculture, and this ability could be blocked by inhibition of CXCR4 signaling. The ASC were found to be nontumorigenic in vitro using a soft agar assay, as well as in vivo, utilizing 2 prostate cancer xenograft models. The ASC-MDA7 only reduced tumor growth in the TRAMP-C2-Ras (TC2Ras) prostate cancer model. The ASC-PEDF, however, reduced growth in both the TC2Ras and the PC3 highly aggressive prostate cancer models, and it was able to completely prevent prostate tumor establishment in vivo. In conclusion, ASC expressing PEDF and MDA7 could effectively reduce prostate tumor growth in vivo, suggesting ASC-cytokine therapies might have translational applications, especially the PEDF modality.

title: Irradiation Enhances the Tumor Tropism and Therapeutic Potential of Tumor Necrosis Factor‐Related Apoptosis‐Inducing Ligand‐Secreting Human Umbilical Cord Blood‐Derived Mesenchymal Stem Cells in Glioma Therapy
abstract: Irradiation is a standard therapy for gliomas and many other cancers. Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is one of the most promising candidates for cancer gene therapy. Here, we show that tumor irradiation enhances the tumor tropism of human umbilical cord blood‐derived mesenchymal stem cells (UCB‐MSCs) and the therapeutic effect of TRAIL delivered by UCB‐MSCs. The sequential treatment with irradiation followed by TRAIL‐secreting UCB‐MSCs (MSC‐TRAIL) synergistically enhanced apoptosis in either TRAIL‐sensitive or TRAIL‐resistant glioma cells by upregulating the death receptor 5 and by inducing caspase activation. Migration assays showed greater MSC migration toward irradiated glioma cells and the tumor site in glioma‐bearing mice compared with unirradiated tumors. Irradiated glioma cells had increased expression of interleukin‐8 (IL‐8), which leads to the upregulation of the IL‐8 receptor on MSCs. This upregulation, which is involved in the migratory capacity of UCB‐MSCs, was confirmed by siRNA inhibition and an antibody‐neutralizing assay. In vivo survival experiments in orthotopic xenografted mice showed that MSC‐based TRAIL gene delivery to irradiated tumors had greater therapeutic efficacy than a single treatment. These results suggest that clinically relevant tumor irradiation increases the therapeutic efficacy of MSC‐TRAIL by increasing tropism of MSCs and TRAIL‐induced apoptosis, which may be a more useful strategy for cancer gene therapy. STEM CELLS 2010;28:2217–2228

title: Neo-organoid of marrow mesenchymal stromal cells secreting interleukin-12 for breast cancer therapy.
abstract: Bone marrow-derived mesenchymal stromal cells (MSCs), beneficial for regenerative medicine applications due to their wide differentiation capabilities, also hold promise as cellular vehicles for the delivery of therapeutic plasma-soluble gene products due to their ease of handling, expansion, and genetic engineering. We hypothesized that MSCs, gene enhanced to express interleukin-12 (IL-12) and then embedded in a matrix, may act as an anticancer neo-organoid when delivered s.c. in autologous/syngeneic hosts. We performed such experiments in mice and noted that primary murine MSCs retrovirally engineered to secrete murine IL-12 can significantly interfere with growth of 4T1 breast cancer cells in vivo, with a more substantial anticancer action achieved when these cells are embedded in a matrix. Plasma of mice that received the IL-12 MSC-containing neo-organoids showed increased levels of IL-12 and IFN-gamma. Histopathologic analysis revealed less tumor cells in implants of 4T1 cells with IL-12 MSCs, and the presence of necrotic tumor islets and necrotic capillaries, suggesting antiangiogenesis. We also showed that the anticancer effect exerted by the IL-12 MSCs is immune mediated because it is absent in immunodeficient mice, is not due to systemic IL-12 delivery, and also occurs in a B16 melanoma model. This study therefore establishes the feasibility of using gene-enhanced MSCs in a cell-based neo-organoid approach for cancer treatment.

title: Intratracheal Delivery of CX3CL1-Expressing Mesenchymal Stem Cells to Multiple Lung Tumors
abstract: The lung is one of the organs to which cancers from solid tumors frequently metastasize. Multiple tumors in the lung are usually treated by systemic chemotherapy because of the lack of efficient methods of targeting antitumor agents to the lung. Although intratracheal administration is an ideal route for targeting multiple lung tumors, antitumor agents are often harmful to the organ or induce inflammation. Mesenchymal stem cells (MSCs), nonhematopoietic stem cells capable of differentiating into various mesoderm-type cells, have a propensity to migrate to and proliferate in tumor tissues after systemic administration. We intratracheally injected MSCs expressing CX3CL1 (MSC/RGDFKN) into the lung of lung tumor-bearing mice with multiple metastases of C26 or Lewis lung carcinoma (LLC). Antitumor effects were evaluated by counting the number of lung metastases and survival. We demonstrated the tropism of mouse MSCs to lung tumor tissues after intratracheal administration of GFP-positive MSCs. Intratracheal injection of MSC/RGDFKN strongly inhibited growth of lung metastases of C26 or LLC, and thus prolonged survival. Intratracheal injection of MSC/RGDFKN did not induce an inflammatory reaction in the lung. These results suggest that MSCs expressing antitumor agents can be delivered intratracheally into multiple lung tumor tissues without causing inflammation.

title: Therapeutic effect of genetically engineered mesenchymal stem cells in rat experimental leptomeningeal glioma model.
abstract: Disseminating disease of high grade gliomas is difficult to treat. We examined the therapeutic effect of intrathecal administration of mesenchymal stem cells transduced with herpes simplex virus-thymidine kinase gene (MSCtk) followed by systemic ganciclovir (GCV) administration in rat experimental leptomeningeal glioma model. First, to examine in vivo bystander effect, rats were intrathecally co-injected with a mixture of MSCtk and C6 cells and then, intraperitoneally administered with GCV or saline for 10days (co-injection model). Next, to examine the therapeutic effect of MSCtk/GCV therapy, MSCtk cells were intrathecally administered 1day after C6 injection and then, GCV or saline was administered (treatment model). GCV administration significantly reduced tumor size on day 14 both in the co-injection model (0.41+/-0.22 vs. 3.10+/-0.97mm(2), p<0.01) and in the treatment model (0.73+/-.29 vs. 2.84+/-0.82mm(2), p<0.01). Survival was also significantly prolonged in GCV group both in the co-injection model (29.2+/-3.3 vs. 18.8+/-0.8days, p<0.001) and in the treatment model (21.5+/-1.5 vs. 17.2+/-0.5days, p<0.001). This study provided a novel treatment strategy for leptomeningeal glioma dissemination using intrathecal MSCtk injection followed by systemic GCV administration.

title: Anti-angiogenesis therapy based on the bone marrow-derived stromal cells genetically engineered to express sFlt-1 in mouse tumor model
abstract: BackgroundBone marrow-derived stromal cells (BMSCs) are important for development, tissue cell replenishment, and wound healing in physiological and pathological conditions. BMSCs were found to preferably reach sites undergoing the process of cell proliferation, such as wound and tumor, suggesting that BMSCs may be used as a vehicle for gene therapy of tumor.MethodsMouse BMSCs were loaded with recombinant adenoviruses which express soluble Vascular Endothelial Growth Factor Receptor-1 (sFlt-1). The anti-angiogenesis of sFlt-1 in BMSCs was determined using endothelial cells proliferation inhibition assay and alginate encapsulation assay. The anti-tumor effects of BMSCs expressing sFlt-1 through tail-vein infusion were evaluated in two mouse tumor metastases models.ResultsBMSCs genetically modified with Adv-GFP-sFlt-1 could effectively express and secret sFlt-1. BMSCs loaded with sFlt-1 gene could preferentially home to tumor loci and decrease lung metastases and prolong lifespan in mouse tumor model through inducing anti-angiogenesis and apoptosis in tumors.ConclusionWe demonstrated that BMSCs might be employed as a promising vehicle for tumor gene therapy which can effectively not only improve the concentration of anticancer therapeutics in tumors, but also modify the tumor microenvironment.

title: Retroviral vector‐producing mesenchymal stem cells for targeted suicide cancer gene therapy
abstract: Mesenchymal stem cells (MSCs) are a promising vehicle for targeted cancer gene therapy because of their potential of tumor tropism. For efficient therapeutic application, we developed retroviral vector‐producing MSCs that enhance tumor transduction via progeny vector production.

title: Bystander killing of malignant glioma by bone marrow-derived tumor-infiltrating progenitor cells expressing a suicide gene.
abstract: Adult stem cells are promising cellular vehicles for therapy of malignant gliomas as they have the ability to migrate into these tumors and even track infiltrating tumor cells. However, their clinical use is limited by a low passaging capacity that impedes large-scale production. In the present study, a bone marrow-derived, highly proliferative subpopulation of mesenchymal stem cells (MSCs)-here termed bone marrow-derived tumor-infiltrating cells (BM-TICs)-was genetically modified for the treatment of malignant glioma. Upon injection into the tumor or the vicinity of the tumor, BM-TICs infiltrated solid parts as well as the border of rat 9L glioma. After intra-tumoral injection, BM-TICs expressing the thymidine kinase of herpes simplex virus (HSV-tk) and enhanced green fluorescent protein (BM-TIC-tk-GFP) were detected by non-invasive positron emission tomography (PET) using the tracer 9-[4-[(18)F]fluoro-3-hydroxymethyl)butyl]guanine ([(18)F]FHBG). A therapeutic effect was demonstrated in vitro and in vivo by BM-TICs expressing HSV-tk through bystander-mediated glioma cell killing. Therapeutic efficacy was monitored by PET as well as by magnetic resonance imaging (MRI) and strongly correlated with histological analysis. In conclusion, BM-TICs expressing a suicide gene were highly effective in the treatment of malignant glioma in a rat model and therefore hold great potential for the therapy of malignant brain tumors in humans.

title: Use of genetically engineered bone marrow-derived mesenchymal stem cells for glioma gene therapy.
abstract: In our previous study, we successfully treated an established C6 brain tumor using neural stem cells transduced with the herpes simplex virus-thymidine kinase gene (HSVtk) and ganciclovir in the rat. In the present study, we investigated the use of mesenchymal stem cells (MSCs), obtained from adult rats and transduced with HSVtk (MSCtk cells), instead of neural stem cells because MSCs are much easier to obtain from the adult subjects. Those cells were used for in vitro co-culture study and in vivo co-implantation study with C6 rat glioma cells to examine bystander tumoricidal effect, which revealed a sufficient bystander effect and only 1/32 MSCtk cells were needed for complete tumor eradication. In vitro bystander effect was also observed in a real-time fashion using a culture microscope and it was shown that only tumor cells that had contact with MSCtk cells died. In vivo treatment study of an established C6 brain tumor with an intratumoral injection of MSCtk cells followed by systemic ganciclovir administration demonstrated a significant reduction of the tumor size and a significant survival prolongation. The treatment strategy using MSCtk and ganciclovir (MSCtk therapy) is more feasible and practical for clinical application than the method using neural stem cells.

title: Mesenchymal stem cells as a gene therapy carrier for treatment of fibrosarcoma.
abstract: Cell-based gene therapy is an alternative to viral and non-viral gene therapy. Emerging evidence suggests that mesenchymal stem cells (MSC) are able to migrate to sites of tissue injury and have immunosuppressive properties that may be useful in targeted gene therapy for sustained specific tissue engraftment.



In this study, we injected intravenously (i.v.) 1x10(6) MSC, isolated from green fluorescent protein (GFP) transgenic rats, into Rif-1 fibrosarcoma-bearing C3H/HeN mice. The MSC had been infected using a lentiviral vector to express stably the luciferase reporter gene (MSC-GFP-luci). An in vivo imaging system (IVIS 200) and Western blotting techniques were used to detect the distribution of MSC-GFP-luci in tumor-bearing animals.



We observed that xenogenic MSC selectively migrated to the tumor site, proliferated and expressed the exogenous gene in subcutaneous fibrosarcoma transplants. No MSC distribution was detected in other organs, such as the liver, spleen, colon and kidney. We further showed that the FGF2/FGFR pathways may play a role in the directional movement of MSC to the Rif-1 fibrosarcoma. We performed in vitro co-culture and in vivo tumor growth analysis, showing that MSC did not affect the proliferation of Rif-1 cells and fibrosarcoma growth compared with an untreated control group. Finally, we demonstrated that the xenogenic MSC stably expressing inducible nitric oxide synthase (iNOS) protein transferred by a lentivirus-based system had a significant inhibitory effect on the growth of Rif-1 tumors compared with MSC alone and the non-treatment control group.



iNOS delivered by genetically modified iNOS-MSC showed a significant anti-tumor effect both in vitro and in vivo. MSC may be used as a target gene delivery vehicle for the treatment of fibrosarcoma and other tumors.

title: Sarcoma Derived from Cultured Mesenchymal Stem Cells
abstract: To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole‐body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain‐specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposon‐related genetic abnormality, and p53 was not overexpressed. Infusion of MSC‐derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures.

title: Accumulated Chromosomal Instability in Murine Bone Marrow Mesenchymal Stem Cells Leads to Malignant Transformation
abstract: Despite recent emerging evidence suggesting that cancer stem cells subsist in a variety of tumors, it is not yet fully elucidated whether postnatal stem cells are directly involved in tumorigenesis. We used murine bone marrow–derived mesenchymal stem cells (BMMSCs) as a model to test a hypothesis that tumorigenesis may originate from spontaneous mutation of stem cells. In this study, we demonstrated that murine BMMSCs, after numerous passages, obtained unlimited population doublings and proceeded to a malignant transformation state, resulting in fibrosarcoma formation in vivo. Transformed BMMSCs colonized to multiple organs when delivered systemically through the tail vein. Fibrosarcoma cells formed by transformed BMMSCs contained cancer progenitors, which were capable of generating colony clusters in vitro and fibrosarcoma in vivo by the second administration. The mechanism by which BMMSCs transformed to malignant cells was associated with accumulated chromosomal abnormalities, gradual elevation in telomerase activity, and increased c‐myc expression. Moreover, BMMSCs and their transformed counterpart, fibrosarcoma‐forming cells, demonstrated different sensitivity to anti‐cancer drugs. BMMSCs/fibrosarcoma transformation system may provide an ideal system to elucidate the mechanism of how stem cells become cancer cells and to screen anti‐sarcoma drugs.

title: Spontaneous expression of embryonic factors and p53 point mutations in aged mesenchymal stem cells: a model of age-related tumorigenesis in mice.
abstract: Aging is the single most common risk factor for cancer. Peripheral and marrow-derived stem cells are long lived and are candidate cells for the cancer-initiating cell. Repeated rounds of replication are likely required for accumulation of the necessary genetic mutations. Based on the facts that mesenchymal stem cells (MSC) transform with higher frequency than other cell types, and tumors in aged C57BL/6 mice are frequently fibrosarcomas, we used a genetically tagged bone marrow (BM) transplant model to show that aged mice develop MSC-derived fibrosarcomas. We further show that, with aging, MSCs spontaneously transform in culture and, when placed into our mouse model, recapitulated the naturally occurring fibrosarcomas of the aged mice with gene expression changes and p53 mutation similar to the in vivo model. Spontaneously transformed MSCs contribute directly to the tumor, tumor vasculature, and tumor adipose tissue, recruit additional host BM-derived cells (BMDC) to the area, and fuse with the host BMDC. Unfused transformed MSCs act as the cancer stem cell and are able to form tumors in successive mice, whereas fusion restores a nonmalignant phenotype. These data suggest that MSCs may play a key role in age-related tumors, and fusion with host cells restores a nonmalignant phenotype, thereby providing a mechanism for regulating tumor cell activity.

title: Chromosomal instability of murine adipose tissue-derived mesenchymal stem cells in long-term culture and development of cloned embryos.
abstract: Mice are the most commonly used laboratory animals for research, and some mouse stem cells, such as induced pluripotent stem cells, embryonic stem cells, and mesenchymal stem cells (MSCs), are also widely used in basic research. It is thus important to know if these stem cells maintain their genomic stability when cultured. Murine bone marrow-derived mesenchymal stem cells (BMSCs) appear to undergo spontaneous transformation in vitro. Murine adipose tissue-derived mesenchymal stem cells (ADSCs), like BMSCs, have the potential to differentiate into multiple lineages. In this study, we used G-banding, induction of multiple-lineage differentiation, flow cytometry, and nuclear transfer (NT), and found that murine ADSCs also displayed chromosomal instability in long-term culture. Furthermore, we performed NT using murine ADSCs to study the nuclear reprogramming ability of undifferentiated adult stem cells and to find a new efficient donor for NT. Using the stem cells did not increase the percentage of NT embryos that developed to the morula/blastocyst stage, compared with cloned embryos from cumulus cells. This may be because the stem cells displayed chromosomal instability. This is the first reported study of the use of ADSCs for NT in mice. ADSCs could provide an alternative donor cell type for NT in other species, with the advantages of easy harvesting involving little or no pain or trauma.

title: Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study
abstract: Severe graft-versus-host disease (GVHD) is a life-threatening complication after allogeneic transplantation with haemopoietic stem cells. Mesenchymal stem cells modulate immune responses in vitro and in vivo. We aimed to assess whether mesenchymal stem cells could ameliorate GVHD after haemopoietic-stem-cell transplantation.



Patients with steroid-resistant, severe, acute GVHD were treated with mesenchymal stem cells, derived with the European Group for Blood and Marrow Transplantation ex-vivo expansion procedure, in a multicentre, phase II experimental study. We recorded response, transplantation-related deaths, and other adverse events for up to 60 months' follow-up from infusion of the cells.



Between October, 2001, and January, 2007, 55 patients were treated. The median dose of bone-marrow derived mesenchymal stem cells was 1.4x10(6) (min-max range 0.4-9x10(6)) cells per kg bodyweight. 27 patients received one dose, 22 received two doses, and six three to five doses of cells obtained from HLA-identical sibling donors (n=5), haploidentical donors (n=18), and third-party HLA-mismatched donors (n=69). 30 patients had a complete response and nine showed improvement. No patients had side-effects during or immediately after infusions of mesenchymal stem cells. Response rate was not related to donor HLA-match. Three patients had recurrent malignant disease and one developed de-novo acute myeloid leukaemia of recipient origin. Complete responders had lower transplantation-related mortality 1 year after infusion than did patients with partial or no response (11 [37%] of 30 vs 18 [72%] of 25; p=0.002) and higher overall survival 2 years after haemopoietic-stem-cell transplantation (16 [53%] of 30 vs four [16%] of 25; p=0.018).



Infusion of mesenchymal stem cells expanded in vitro, irrespective of the donor, might be an effective therapy for patients with steroid-resistant, acute GVHD.

title: The Immunogenicity of Human Adipose‐Derived Cells: Temporal Changes In Vitro
abstract: Regenerative medical techniques will require an abundant source of human adult stem cells that can be readily available at the point of care. The ability to use unmatched allogeneic stem cells will help achieve this goal. Since adipose tissue represents an untapped reservoir of human cells, we have compared the immunogenic properties of freshly isolated, collagenase‐digested human adipose tissue‐derived stromal vascular fraction cells (SVFs) relative to passaged, plastic‐adherent adipose‐derived stem cells (ASCs). Parallel studies have shown that adherence to plastic and subsequent expansion of human adipose‐derived cells selects for a relatively homogeneous cell population based on immunophenotype. Consistent with these findings, the presence of hematopoietic‐associated markers (CD11a, CD14, CD45, CD86, and histocompatible locus antigen‐DR [HLA‐DR]) detected on the heterogeneous SVF cell population decreased upon subsequent passage of the ASCs. In mixed lymphocyte reactions (MLRs), SVFs, and early passage ASCs stimulated proliferation by allogeneic responder T cells. In contrast, the ASCs beyond passage P1 failed to elicit a response from T cells. Indeed, late passage ASCs actually suppressed the MLR response. Although these results support the feasibility of allogeneic human ASC transplantation, confirmatory in vivo animal studies will be required.

title: Human mesenchymal stem cells (hMSCs) expressing truncated soluble vascular endothelial growth factor receptor (tsFlk‐1) following lentiviral‐mediated gene transfer inhibit growth of Burkitt's lymphoma in a murine model
abstract: Efficient gene transfer to bone marrow derived mesenchymal stem cells (MSC) would provide an important opportunity to express potent anticancer agents in the tumour microenvironment because of their contribution to the tumour stroma.

title: Hybrid herpes simplex virus/Epstein–Barr virus amplicon viral vectors confer enhanced transgene expression in primary human tumors and human bone marrow‐derived mesenchymal stem cells
abstract: Herpes simplex virus type‐1 (HSV‐1) amplicon vectors are attractive tools for gene transfer because of their large DNA insert capacity, their broad host range of vector transduction and a minimal immune response as a result of the absence of helper viruses during viral packaging. However, the transient gene expression remains a challenge for the translation of HSV‐1 amplicon based therapeutic strategies to a clinical setting. Although oriP/EBV nuclear antigen (EBNA)‐1 elements of Epstein–Barr virus (EBV) have been successfully employed to achieve prolonged transgene expression, little is known about the stability of the EBNA‐1 elements in the context of HSV‐1 amplicon viral vectors.

title: Comparative analysis of transposable element vector systems in human cells.
abstract: Transposon-based gene vectors have become indispensable tools in vertebrate genetics for applications ranging from insertional mutagenesis and transgenesis in model species to gene therapy in humans. The transposon toolkit is expanding, but a careful, side-by-side characterization of the diverse transposon systems has been lacking. Here we compared the Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposons with respect to overall activity, overproduction inhibition (OPI), target site selection, transgene copy number as well as long-term expression in human cells. SB was the most efficient system under conditions where the availability of the transposon DNA is limiting the transposition reaction including hard-to-transfect hematopoietic stem/progenitor cells (HSCs), and the most sensitive to OPI, underpinning the need for careful optimization of the transposon components. SB and PB were about equally active, and both more efficient than Tol2, under nonrestrictive conditions. All three systems provided long-term transgene expression in human cells with minimal signs of silencing. Indeed, mapping of Tol2 insertion sites revealed significant underrepresentation within chromosomal regions with H3K27me3 histone marks typically associated with transcriptionally repressed heterochromatin. SB, Tol2, and PB constitute complementary research tools for gene transfer in mammalian cells with important implications for fundamental and translational research.

title: The Sleeping Beauty transposon system: a non-viral vector for gene therapy.
abstract: Over the past decade, the Sleeping Beauty (SB) transposon system has been developed as the leading non-viral vector for gene therapy. This vector combines the advantages of viruses and naked DNA. Here we review progress over the last 2 years in vector design, methods of delivery and safety that have supported its use in the clinic. Currently, the SB vector has been validated for ex vivo gene delivery to stem cells, including T-cells for the treatment of lymphoma. Progress in delivery of SB transposons to liver for treatment of various systemic diseases, such as hemophilia and mucopolysaccharidoses types I and VII, has encountered some problems, but even here progress is being made.

title: Human adult CD34- progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4.
abstract: The homing and tissue-specific recruitment of bone marrow-derived progenitor cells is a major issue in stem cell research and therapy. Chemokine biology plays a central role in the homing and trafficking of leukocytes. Here we show functional expression of the chemokine receptors CCR1, CCR4, CCR7, CCR10, and CXCR5 on primary isolates of CD34- mesenchymal progenitor cells as well as immortalized mesenchymal stem cell (MSC) lines. Although mRNA expression of CXCR4 was detected in both primary cells and immortalized clones, the receptor was not expressed on the cell surface. On the basis of this expression profile, the MSC could potentially home to secondary lymphatic organs (CCR7, CXCR5), skin (CCR4, CCR10), small intestine (CCR10), and salivary glands (CCR10). To study tissue-specific homing, murine CD34- MSC lines showing concordant chemokine receptor expression were either transiently labeled with CMFDA, or were stably transfected with green fluorescent protein (GFP) expression plasmids. The MSC were then injected into syngeneic healthy mice, and the distribution of the cells determined. The injected cells efficiently homed to spleen, thymus, and lymph nodes. In addition, cells were found in the mucosa of the small intestine, skin, and salivary gland. No significant recruitment to bone marrow, liver, or kidney was seen. Chemokine biology may play an important role in the homeostasis and potentially tissue recruitment of early adult progenitor cells.

title: Mesenchymal Stem Cells Exhibit Firm Adhesion, Crawling, Spreading and Transmigration across Aortic Endothelial Cells: Effects of Chemokines and Shear
abstract: Mesenchymal stem cells (MSCs) have anti-inflammatory and immunosuppressive properties and may be useful in the therapy of diseases such as arteriosclerosis. MSCs have some ability to traffic into inflamed tissues, however to exploit this therapeutically their migratory mechanisms need to be elucidated. This study examines the interaction of murine MSCs (mMSCs) with, and their migration across, murine aortic endothelial cells (MAECs), and the effects of chemokines and shear stress. The interaction of mMSCs with MAECs was examined under physiological flow conditions. mMSCs showed lack of interaction with MAECs under continuous flow. However, when the flow was stopped (for 10min) and then started, mMSCs adhered and crawled on the endothelial surface, extending fine microvillous processes (filopodia). They then spread extending pseudopodia in multiple directions. CXCL9 significantly enhanced the percentage of mMSCs adhering, crawling and spreading and shear forces markedly stimulated crawling and spreading. CXCL9, CXCL16, CCL20 and CCL25 significantly enhanced transendothelial migration across MAECs. The transmigrated mMSCs had down-regulated receptors CXCR3, CXCR6, CCR6 and CCR9. This study furthers the knowledge of MSC transendothelial migration and the effects of chemokines and shear stress which is of relevance to inflammatory diseases such as arteriosclerosis.

title: Human Bone Marrow Stromal Cells Express a Distinct Set of Biologically Functional Chemokine Receptors
abstract: Stromal cells isolated from bone marrow (BMSCs), often referred to as mesenchymal stem cells, are currently under investigation for a variety of therapeutic applications. However, limited data are available regarding receptors that can influence their homing to and positioning within the bone marrow. In the present study, we found that second passage BMSCs express a unique set of chemokine receptors: three CC chemokine receptors (CCR1, CCR7, and CCR9) and three CXC chemokine receptors (CXCR4, CXCR5, and CXCR6). BMSCs cultured in serum‐free medium secrete several chemokine ligands (CCL2, CCL4, CCL5, CCL20, CXCL12, CXCL8, and CX3CL1). The surface‐expressed chemokine receptors were functional by several criteria. Stimulation of BMSCs with chemokine ligands triggers phosphorylation of the mitogen‐activated protein kinase (e.g., extracellular signal–related kinase [ERK]‐1 and ERK‐2) and focal adhesion kinase signaling pathways. In addition, CXCL12 selectively activates signal transducer and activator of transcription (STAT)‐5 whereas CCL5 activates STAT‐1. In cell biologic assays, all of the chemokines tested stimulate chemotaxis of BMSCs, and CXCL12 induces cytoskeleton F‐actin polymerization. Studies of culture‐expanded BMSCs, for example, 12–16 passages, indicate loss of surface expression of all chemokine receptors and lack of chemotactic response to chemokines. The loss in chemokine receptor expression is accompanied by a decrease in expression of adhesion molecules (ICAM‐1, ICAM‐2, and vascular cell adhesion molecule 1) and CD157, while expression of CD90 and CD105 is maintained. The change in BMSC phenotype is associated with slowing of cell growth and increased spontaneous apoptosis. These findings suggest that several chemokine axes may operate in BMSC biology and may be important parameters in the validation of cultured BMSCs intended for cell therapy.

title: The In Vitro Migration Capacity of Human Bone Marrow Mesenchymal Stem Cells: Comparison of Chemokine and Growth Factor Chemotactic Activities
abstract: Adult bone marrow (BM)‐derived stem cells, including hematopoietic stem cells (HSCs) and MSCs, represent an important source of cells for the repair of a number of damaged tissues. In contrast to HSCs, the soluble factors able to induce MSC migration have not been extensively studied. In the present work, we compared the in vitro migration capacity of human BM‐derived MSCs, preincubated or not with the inflammatory cytokines interleukin 1β (IL1β) and tumor necrosis factor α (TNFα), in response to 16 growth factors (GFs) and chemokines. We show that BM MSCs migrate in response to many chemotactic factors. The GFs platelet‐derived growth factor‐AB (PDGF‐AB) and insulin‐like growth factor 1 (IGF‐1) are the most potent, whereas the chemokines RANTES, macrophage‐derived chemokine (MDC), and stromal‐derived factor‐1 (SDF‐1) have limited effect. Remarkably, preincubation with TNFα leads to increased MSC migration toward chemokines, whereas migration toward most GFs is unchanged. Consistent with these results, BM MSCs express the tyrosine kinase receptors PDGF‐receptor (R) α, PDGF‐Rβ, and IGF‐R, as well as the RANTES and MDC receptors CCR2, CCR3, and CCR4 and the SDF‐1 receptor CXCR4. TNFα increases CCR2, CCR3, and CCR4 expression (as opposed to that of CXCR4), together with RANTES membrane binding. These data indicate that the migration capacity of BM MSCs is under the control of a large range of receptor tyrosine kinase GFs and CC and CXC chemokines. Most chemokines are more effective on TNFα‐primed cells. Our results suggest that the mobilization of MSCs and their subsequent homing to injured tissues may depend on the systemic and local inflammatory state.

title: Towards in situ tissue repair: Human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2
abstract: The recruitment of bone marrow CD34− mesenchymal stem‐ and progenitor cells (MSC) and their subsequent differentiation into distinct tissues is the precondition for in situ tissue engineering. The objective of this study was to determine the entire chemokine receptor expression profile of human MSC and to investigate their chemotactic response to the selected chemokines CCL2, CXCL8 and CXCL12. Human MSC were isolated from iliac crest bone marrow aspirates and showed a homogeneous population presenting a typical MSC‐related cell surface antigen profile (CD14−, CD34−, CD44+, CD45−, CD166+, SH‐2+). The expression profile of all 18 chemokine receptors was determined by real‐time PCR and immunohistochemistry. Both methods consistently demonstrated that MSC express CC, CXC, C and CX3C receptors. Gene expression and immunohistochemical analysis documented that MSC express chemokine receptors CCR2, CCR8, CXCR1, CXCR2 and CXCR3. A dose‐dependent chemotactic activity of CXCR4 and CXCR1/CXCR2 ligands CXCL12 and CXCL8 (interleukin‐8) was demonstrated using a 96‐well chemotaxis assay. In contrast, the CCR2 ligand CCL2 (monocyte chemoattractant protein‐1, MCP‐1) did not recruited human MSC. In conclusion, we report that the chemokine receptor expression profile of human MSC is much broader than known before. Furthermore, for the first time, we demonstrate that human MSC migrate upon stimulation with CXCL8 but not CCL2. In combination with already known data on MSC recruitment and differentiation these are promising results towards in situ regenerative medicine approaches based on guiding of MSC to sites of degenerated tissues.  2007.

title: Human mesenchymal stem cells overexpressing pigment epithelium-derived factor inhibit hepatocellular carcinoma in nude mice
abstract: The poor outcome of cancer gene therapy in clinical trials relates in part to insufficient gene delivery to tumor sites. Mesenchymal stem cells (MSCs) represent a new tool for the delivery of therapeutic agents to tumor cells. This study used an orthotopic nude mice model of hepatocellular carcinoma (HCC) to evaluate the potential of genetically modified human MSCs (hMSCs), to function as an effective delivery vehicle for therapeutic genes. hMSCs derived from the bone marrow were efficiently engineered to express human pigment epithelium-derived factor (PEDF) by lentiviral transduction, then tested in vitro for high-level expression and bioactivity of the transgenic protein. The preferential homing of hMSCs toward HCC was confirmed by in vitro and in vivo migration assays. in vivo efficacy experiments showed that intravenous (i.v.) injection of PEDF-expressing hMSCs significantly suppressed both the growth of primary liver tumors and the development of pulmonary metastases. Moreover, hMSCs-based PEDF gene delivery moderately increased the systemic levels of human PEDF. Immunohistochemistry of primary liver tumors demonstrated lower microvessel density in mice treated with hMSCs-PEDF than in control mice. This is the first study to show the potential of hMSCs as an effective delivery vehicle for therapeutic genes in the treatment of HCC.

title: VEGF expression by mesenchymal stem cells contributes to angiogenesis in pancreatic carcinoma
abstract: Little is known about the factors that enable the mobilisation of human mesenchymal stem cells (MSC) from the bone marrow into the blood stream and their recruitment to and retention in the tumour. We found specific migration of MSC towards growth factors present in pancreatic tumours, such as PDGF, EGF, VEGF and specific inhibitors Glivec, Erbitux and Avastin interfered with migration. Within a few hours, MSC migrated into spheroids consisting of pancreatic cancer cells, fibroblasts and endothelial cells as measured by time-lapse microscopy. Supernatant from subconfluent MSC increased sprouting of HUVEC due to VEGF production by MSC itself as demonstrated by RT-PCR and ELISA. Only few MSCs were differentiated into endothelial cells in vitro, whereas in vivo differentiation was not observed. Lentiviral GFP-marked MSCs, injected in nude mice xenografted with orthotopic pancreatic tumours, preferentially migrated into the tumours as observed by FACS analysis of green fluorescent cells. By immunofluorescence and intravital microscopic studies, we found the interaction of MSC with the endothelium of blood vessels. Mesenchymal stem cells supported tumour angiogenesis in vivo, that is CD31+ vessel density was increased after the transfer of MSC compared with siVEGF-MSC. Our data demonstrate the migration of MSC toward tumour vessels and suggest a supportive role in angiogenesis.

title: Image-guided, tumor stroma-targeted 131I therapy of hepatocellular cancer after systemic mesenchymal stem cell-mediated NIS gene delivery.
abstract: Due to its dual role as reporter and therapy gene, the sodium iodide symporter (NIS) allows noninvasive imaging of functional NIS expression by (123)I-scintigraphy or (124)I-PET imaging before the application of a therapeutic dose of (131)I. NIS expression provides a novel mechanism for the evaluation of mesenchymal stem cells (MSCs) as gene delivery vehicles for tumor therapy. In the current study, we stably transfected bone marrow-derived CD34(-) MSCs with NIS cDNA (NIS-MSC), which revealed high levels of functional NIS protein expression. In mixed populations of NIS-MSCs and hepatocellular cancer (HCC) cells, clonogenic assays showed a 55% reduction of HCC cell survival after (131)I application. We then investigated body distribution of NIS-MSCs by (123)I-scintigraphy and (124)I-PET imaging following intravenous (i.v.) injection of NIS-MSCs in a HCC xenograft mouse model demonstrating active MSC recruitment into the tumor stroma which was confirmed by immunohistochemistry and ex vivo γ-counter analysis. Three cycles of systemic MSC-mediated NIS gene delivery followed by (131)I application resulted in a significant delay in tumor growth. Our results demonstrate tumor-specific accumulation and therapeutic efficacy of radioiodine after MSC-mediated NIS gene delivery in HCC tumors, opening the prospect of NIS-mediated radionuclide therapy of metastatic cancer using MSCs as gene delivery vehicles.

title: Selective Targeting of Genetically Engineered Mesenchymal Stem Cells to Tumor Stroma Microenvironments Using Tissue-Specific Suicide Gene Expression Suppresses Growth of Hepatocellular Carcinoma
abstract: The use of engineered mesenchymal stem cells (MSCs) as therapeutic vehicles for the treatment of experimental pancreatic and breast cancer has been previously demonstrated. The potential application of MSCs for the treatment of hepatocellular carcinoma (HCC) has been controversial. The general approach uses engineered MSCs to target different aspects of tumor biology, including angiogenesis or the fibroblast-like stromal compartment, through the use of tissue-specific expression of therapeutic transgenes. The aim of the present study was (1) to evaluate the effect of exogenously added MSCs on the growth of HCC and (2) the establishment of an MSC-based suicide gene therapy for experimental HCC. Mesenchymal stem cells were isolated from bone marrow of C57/Bl6 p53−/− mice. The cells were injected into mice with HCC xenografts and the effect on tumor proliferation and angiogenesis was evaluated. The cells were then stably transfected with red fluorescent protein (RFP) or Herpes simplex virus thymidine kinase (HSV-Tk) gene under control of the Tie2 promoter/enhancer or the CCL5 promoter. Mesenchymal stem cells were injected intravenously into mice with orthotopically growing xenografts of HCC and treated with ganciclovir (GCV). Ex vivo examination of hepatic tumors revealed tumor-specific recruitment, enhanced tumor growth, and increased microvessel density after nontherapeutic MSC injections. After their homing to the hepatic xenografts, engineered MSCs demonstrated activation of the Tie2 or CCL5 promoter as shown by RFP expression. Application of CCL5/HSV-TK transfected MSCs in combination with GCV significantly reduced tumor growth by 56.4% as compared with the control group and by 71.6% as compared with nontherapeutic MSC injections. CCL5/HSV-TK+ transfected MSCs proved more potent in tumor inhibition as compared with Tie2/HSV-TK+ MSCs. Exogenously added MSCs are recruited to growing HCC xenografts with concomitant activation of the CCL5 or Tie2 promoters within the MSCs. Stem cell–mediated introduction of suicide genes into the tumor followed by prodrug administration was effective for treatment of experimental HCC and thus may help fill the existing gap in bridging therapies for patients suffering from advanced HCCs.

title: Autologous Mesenchymal Stem Cell Therapy Delays the Progression of Neurological Deficits in Patients With Multiple System Atrophy
abstract: We evaluated the feasibility and safety of therapy with mesenchymal stem cells (MSCs) through consecutively intra‐arterial and three repeated intravenous injections and compared the long‐term prognosis between MSC‐treated (n=11) and control multiple system atrophy (MSA) patients (n=18). The MSC‐treated patients showed significantly greater improvement on the unified MSA rating scale (UMSARS) than the control patients at all visits throughout the 12‐month study period. Orthostasis in UMSARS I items and cerebellar dysfunction‐related items of UMSARS II items were significantly different in favor of MSC treatment compared to controls. Serial positron emission tomography scan in the MSC‐treated group showed that increased fluorodeoxyglucose uptake from baseline was noted in cerebellum and frontal white matters. No serious adverse effects related to MSC therapy occurred. This study demonstrated that MSC therapy in patients with MSA was safe and delayed the progression of neurological deficits with achievement of functional improvement in the follow‐up period.

title: Orthotopic reconstitution of human small-cell lung carcinoma after intravenous transplantation in SCID mice.
abstract: We have constructed an orthotopically reconstituted model of human small-cell lung carcinoma (SCLC) by intravenous transplantation in severe combined immunodeficient (SCID) mice. Two human SCLC xenografts, H-69 and Lu-130, were disaggregated and injected through the tail vein of SCID mice. Human SCLCs were orthotopically reconstituted with multi-focal lung tumor growth in all SCID mice after intravenous injection of 5 x 10(6) tumor cells per mouse. The heart and liver were also seeded with actively growing SCLC. This orthotopic reconstitution model of human SCLC in SCID mice should be useful for further studies on the biological behavior and treatment of human SCLC.

title: Targeting Tumor Stroma Using Engineered Mesenchymal Stem Cells Reduces the Growth of Pancreatic Carcinoma
abstract: To analyze the efficacy of engineered mesenchymal stem cell based therapy directed towards pancreatic tumor stroma. Mesenchymal stem cells (MSC) are actively recruited to tumor stroma where they enhance tumor growth and metastases. Upregulation of chemotactic cytokine (CCL5) by MSCs within the tumor stroma has been shown to play a central role in this process. Murine MSCs were engineered to express reporter genes or therapeutic genes under control of the CCL5 promoter and adoptively transferred into mice with growing pancreatic tumors. The effect on tumor growth and metastases was then evaluated. MSCs isolated from bone marrow of C57/Bl6 p53−/− mice were stably transfected with red fluorescent protein (RFP), enhanced green fluorescent protein (eGFP), or herpes simplex virus (HSV) thymidine kinase (Tk) gene driven by the RANTES promoter. MSCs were intravenously applied once per week over 3 weeks to mice carrying an orthotopic, syngeneic pancreatic Panc02 tumor. eGFP and RFP signals driven by the CCL5 promoter were detected by fluorescence in treated pancreatic tumor samples. The HSV-Tk therapy group treated intraperitoneal with the prodrug ganciclovir 5 to 7 days after stem cell application lead to a 50% reduction of primary pancreatic tumor growth (P < 0.0003, student t test) and reduced liver metastases (0% vs. 60%). The active homing of MSCs into primary pancreatic tumor stroma and activation of the CCL5 promoter was verified using eGFP- and RFP-reporter genes. In the presence of ganciclovir, HSV-Tk transfected MSCs led to a significant reduction of primary pancreatic tumor growth and incidence of metastases.

title: Safety of intravenous infusion of human adipose tissue-derived mesenchymal stem cells in animals and humans.
abstract: Adipose tissue-derived mesenchymal stem cells (AdMSCs) represent an attractive and ethical cell source for stem cell therapy. With the recent demonstration of MSC homing properties, intravenous applications of MSCs to cell-damaged diseases have increased. In the present study, the toxicity and tumorigenicity of human AdMSCs  were investigated for clinical application.  showed the typical appearance, immunophenotype, and differentiation capacity of MSCs, and were genetically stable at least 12 passages in culture. Cells suspended in physiological saline maintained their MSC properties in a cold storage condition for at least 3 days. To test the toxicity of hAdMSCs, different doses of hAdMSCs were injected intravenously into immunodeficient mice, and the mice were observed for 13 weeks. Even at the highest cell dose (2.5×10(8) cells/kg body weight), the SCID mice were viable and had no side effects. A tumorigenicity test was performed in Balb/c-nu nude mice for 26 weeks. Even at the highest cell dose (2×10(8) MSCs/kg), no evidence of tumor development was found. In a human clinical trial, 8 male patients who had suffered a spinal cord injury >12 months previous were intravenously administered autologous hAdMSCs (4×10(8) cells) one time. None of the patients developed any serious adverse events related to hAdMSC transplantation during the 3-month follow-up. In conclusion, the systemic transplantation of hAdMSCs appears to be safe and does not induce tumor development.

title: Systemic administration of autologous adipose-derived mesenchymal stem cells alleviates hepatic ischemia–reperfusion injury in rats
abstract: Mesenchymal stem cells have previously been shown to offer significant therapeutic benefit in ischemic organ injuries. This study aimed at investigating the therapeutic role of adipose tissue-derived mesenchymal stem cells in hepatic ischemia–reperfusion injury and the underlying mechanisms. Adult male Fisher rats (n = 30) were equally divided into three groups (group 1: Sham-operated normal controls; group 2: Ischemia-reperfusion injury with intravenous fresh culture medium; group 3: Ischemia-reperfusion injury with intravenous adipose tissue-derived mesenchymal stem cells). Ischemia-reperfusion injury was induced by occluding the vascular supplies of left lobe liver for 60 minutes followed by reperfusion for 72 hrs. Adipose tissue-derived mesenchymal stem cells (1.2 × 106) were administered through tail vein immediately after reperfusion and at 6 hrs and 24 hrs after reperfusion in group 3. All animals were sacrificed 72 hrs after reperfusion. Animal laboratory at a medical institute. Histologic features, plasma aspartate aminotransferase, hepatic cytokine profile, oxidative stress, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling were analyzed. Seventy-two hrs after reperfusion, plasma aspartate aminotransferase, hepatic oxidative stress, messenger RNA expressions of tumor necrosis factor-a, transforming growth factor-b, interleukin-1b, interleukin-6, endothelin-1, matrix metalloproteinase-9, plasminogen activator inhibitor-1, Bax and caspase-3, protein expression of intercellular adhesion molecule as well as the number of apoptotic nuclei were significantly increased in group 2 compared with group 3, whereas messenger RNA expressions of endothelial nitric oxide synthase, Bcl-2, interleukin-10, protein expressions of reduced nicotinamide-adenine dinucleotide phosphate:quinone oxidoreductase 1, and heme oxygenase-1 were lower in group 2 than group 3. The results showed that systemic adipose tissue-derived mesenchymal stem cell administration significantly preserved hepatocyte integrity and suppressed inflammatory responses, oxidative stress, and apoptosis in a rodent model of hepatic ischemia–reperfusion injury.

title: Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro promotes hepatic integration in vivo
abstract: The hepatic integration of human adipose tissue derived mesenchymal stem cells (hAT-MSCs) in vivo with or without prior differentiation to hepatocyte-like cells in vitro was investigated.  Cells, isolated either from peritoneal or subcutaneous adipose tissue, expressed mesenchymal stem cell surface markers and featured multiple lineage differentiation. Under conditions favouring hepatocyte differentiation, hAT-MSCs gained hepatocytic functions in vitro including urea formation, glycogen synthesis, cytochrome P450 enzyme activity, and expression of hepatocyte-specific transcripts of carbamoylphosphate synthetase, albumin and cytochrome P450 type 3A4 (CYP3A4). Transgenic expression of green fluorescent protein emerged upon hepatocyte differentiation when driven by the hepatocyte-specific promoter of the cytosolic phosphoenolpyruvate carboxykinase gene but was constitutive from the ubiquitin gene promoter. Human AT-MSCs were transplanted into livers of immunodeficient Pfp/Rag2−/− mice with or without prior hepatocyte differentiation in vitro. Donor-derived human cells engrafted in the mouse host liver predominantly in the periportal region of the liver lobule. They expressed HepPar1 and albumin, typical features of differentiated human hepatocytes, in the otherwise negative mouse liver background. Engraftment was significantly more efficient using hAT-MSCs pre-differentiated to hepatocyte-like cells in vitro as compared with undifferentiated cells.  Pre-differentiation of human MSCs from adipose tissue into hepatocyte-like cells in vitro facilitates long term functional hepatic integration in vivo.

title: Stem cells for hepatic regeneration: the role of adipose tissue derived mesenchymal stem cells.
abstract: Severe hepatic dysfunctions including hepatic cirrhosis and hepatocarcinoma are life-threatening conditions for which effective medical treatments are needed. With the only effective treatment to date being orthotropic liver transplantation, alternative approaches are needed because of the limited number of donors and the possibility of immune-rejection. One alternative is regenerative medicine, which holds promise for the development of a cell-based therapy enabling hepatic regeneration through transplantation of adipose tissue-derived mesenchymal stem cells (AT-MSCs) or hepatocyte-like cells generated from AT-MSCs. When compared with embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, the use of AT-MSCs as regenerative cells would be advantageous in regard to ethical and safety issues since AT-MSCs are somatic cells and have the potential to be used without in vitro culture. These autologous cells are immuno-compatible and exhibit controlled differentiation and multi-functional abilities and do not undergo post-transplantation rejection or unwanted differentiation such as formation of teratomas. AT-MSC-based therapies may provide a novel approach for hepatic regeneration and hepatocyte differentiation and thereby support hepatic function in diseased individuals.

title: Adipose tissue-derived mesenchymal stem cells and hepatic differentiation: old concepts and future perspectives.
abstract: Mesenchymal stem cells (MSCs) are multipotent cells, able to differentiate into elements of the mesodermal lineage. Bone marrow and adipose tissue represent the main sources for MSC isolation. In the last decade, several studies have reported the plasticity of MSCs toward a hepatocyte-like phenotype. The use of MSCs to generate hepatocyte-like cells holds great promises to overcome the scarcity of available organs for transplantation. However, little is known about the molecular pathways involved in lineage cross-differentiation and several issues remain to be answered before MSC application in clinical settings. Aim of this review is to critically analyze the possible sources of MSCs suitable for liver repopulation and the molecular mechanisms underlying MSC hepatic differentiation.

title: Suppression of tumorigenesis by human mesenchymal stem cells in a hepatoma model
abstract: Human mesenchymal stem cells (hMSCs) can home to tumor sites and inhibit the growth of tumor cells. Little is known about the underlying molecular mechanisms that link hMSCs to the targeted inhibition of tumor cells. In this study, we investigated the effects of hMSCs on two human hepatoma cell lines (H7402 and HepG2) using an animal transplantation model, a co-culture system and conditioned media from hMSCs. Animal transplantation studies showed that the latent time for tumor formation was prolonged and that the tumor size was smaller when SCID mice were injected with H7402 cells and an equal number of Z3 hMSCs. When co-cultured with Z3 cells, H7402 cell proliferation decreased, apoptosis increased, and the expression of Bcl-2, c-Myc, proliferating cell nuclear antigen (PCNA) and survivin was downregulated. After treatment with conditioned media derived from Z3 hMSC cultures, H4702 cells showed decreased colony-forming ability and decreased proliferation. Immunoblot analysis showed that β-catenin, Bcl-2, c-Myc, PCNA and survivin expression was downregulated in H7402 and HepG2 cells. Taken together, our findings demonstrate that hMSCs inhibit the malignant phenotypes of the H7402 and HepG2 human liver cancer cell lines, which include proliferation, colony-forming ability and oncogene expression both in vitro and in vivo. Furthermore, our studies provide evidence that the Wnt signaling pathway may have a role in hMSC-mediated targeting and tumor cell inhibition.

title: NF-κB downregulation may be involved the depression of tumor cell proliferation mediated by human mesenchymal stem cells
abstract: It has been reported that stem cells are able to home to tumorigenesis and inhibit the proliferation of tumor cells. The purpose of our study was to demonstrate the molecular mechanism of the inhibitory proliferation of hepatoma cells and breast cancer cells mediated by human mesenchymal stem cells (hMSCs).Methods:The proliferation of H7402 human hepatoma cells and MCF-7 human breast cancer cells was measured by the 5-bromodeoxyuridine (BrdU) incorporation assay and flow cytometry assay after the treatment with conditioned media from hMSCs culture, such as Z3 cells or BMMS-03 cells. The role of NF-κB or the phosphorylation of inhibitor κBα (p-IκBα) in the depression of hepatoma or breast cancer cells treated with conditioned media from Z3 cells or BMMS-03 cells was examined by reporter gene assay, quantitative real-time PCR, and Western blot analysis, respectively.The proliferation of H7402 cells and MCF-7 cells was decreased significantly by the BrdU incorporation assay and flow cytometry assay after treatment. The transcriptional activity and mRNA level of NF-κB were downregulated in the treated cells by reporter gene assay and quantitative real-time PCR in a dose-dependent manner. At the protein level, NF-κB and p-IκBα decreased in the treated cells by Western blot analysis.Conditioned media from hMSCs are able to inhibit the proliferation of tumor cells. NF-κB downregulation is one of reasons for the depression of tumor cell proliferation mediated by hMSCs.

title: Linking Transgene Expression of Engineered Mesenchymal Stem Cells and Angiopoietin-1–induced Differentiation to Target Cancer Angiogenesis
abstract: To specifically target tumor angiogenesis by linking transgene expression of engineered mesenchymal stem cells to angiopoietin-1–induced differentiation. Mesenchymal stem cells (MSCs) have been used to deliver therapeutic genes into solid tumors. These strategies rely on their homing mechanisms only to deliver the therapeutic agent. We engineered murine MSC to express reporter genes or therapeutic genes under the selective control of the Tie2 promoter/enhancer. This approach uses the differentiative potential of MSCs induced by the tumor microenvironment to drive therapeutic gene expression only in the context of angiogenesis. When injected into the peripheral circulation of mice with either, orthotopic pancreatic or spontaneous breast cancer, the engineered MSCs were actively recruited to growing tumor vasculature and induced the selective expression of either reporter red florescent protein or suicide genes [herpes simplex virus-thymidine kinase (TK) gene] when the adoptively transferred MSC developed endothelial-like characteristics. The TK gene product in combination with the prodrug ganciclovir (GCV) produces a potent toxin, which affects replicative cells. The homing of engineered MSC with selective induction of TK in concert with GCV resulted in a toxic tumor-specific environment. The efficacy of this approach was demonstrated by significant reduction in primary tumor growth and prolongation of life in both tumor models. This “Trojan Horse” combined stem cell/gene therapy represents a novel treatment strategy for tailored therapy of solid tumors.

title: Human mesenchymal stem cells inhibit metastasis of a hepatocellular carcinoma model using the MHCC97‐H cell line
abstract: The effects of mesenchymal stem cells (MSC) on the growth and metastasis of human malignancies including hepatocellular carcinoma (HCC) are controversial, and the underlying mechanisms are not yet understood. The aim of this study was to explore the role of MSC in the progression of HCC. We investigated the effect of MSC on in vitro proliferation and invasion and in vivo tumor growth and pulmonary metastasis of MHCC97‐H HCC cells with a high metastatic potential. The mRNA and protein levels of transforming growth factor‐beta 1 (TGFβ1) and MMP, and their association with the effects of MSC on HCC cells were also evaluated. Co‐culture of MHCC97‐H cells with MSC conditioned medium significantly enhanced in vitro proliferation but inhibited invasiveness. Following MSC treatment of a nude mouse model bearing human HCC, the MSC were predominantly located in the HCC tissues. Compared with controls, MSC‐treated mice exhibited significantly larger tumors (3080.51 ± 1234.78 mm3vs 2223.75 ± 1000.60 mm3, P = 0.045), but decreased cellular numbers of lung metastases (49.75 ± 18.86 vs 227.22 ± 74.67, P = 0.046). Expression of TGFβ1 and MMP‐2 was significantly downregulated in the MSC‐treated HCC cells. TGFβ siRNA concurrently downregulated expression of TGFβ and MMP‐2 in HCC cells and blocked the MSC‐induced proliferation and invasiveness of MHCC97‐H cells. The MSC enhanced tumor growth but significantly inhibited the invasiveness and metastasis of HCC, possibly through downregulation of TGFβ1. These findings suggest that MSC could be useful in controlling metastatic recurrence of HCC. ( 2546–2553)

title: Human mesenchymal stem cells play a dual role on tumor cell growth in vitro and in vivo
abstract: Nowadays, some evidences demonstrate that human mesenchymal stem cells (hMSCs) favor tumor growth; however, others show that hMSCs can suppress tumorigenesis and tumor growth. With the indeterminateness of the effect of hMSCs on tumors, we investigated the effect of hMSCs on lung cancer cell line A549 and esophageal cancer cell line Eca‐109 in vitro and in vivo. Our results revealed that hMSCs inhibited the proliferation and invasion of A549 and Eca‐109 cells, arrested tumor cells in the G1 phase of the cell cycle and induced the apoptosis of tumor cells in vitro by using a co‐culture system and the hMSCs‐conditioned medium. However, animal study showed that hMSCs enhanced tumor formation and growth in vivo. Western blotting and immunoprecipitation data showed that the expressions of proliferating cell nuclear antigen (PCNA), Cyclin E, phospho‐retinoblastoma protein (pRb), B‐cell lymphoma/leukemia‐2 (Bcl‐2), Bcl‐xL, and matrix metalloproteinase 2 (MMP‐2) were downregulated and the formation of Cyclin E‐cyclin‐dependent kinase 2 (CDK2) complexes was inhibited in the tumor cells treated with the hMSCs‐conditioned medium. According to the observation of tumor mass and the result of microvessel density (MVD), we found that the promoting role of hMSCs on tumor growth was related with the increase of tumor vessel formation. Our present study suggests that hMSCs have a contradictory effect on tumor cell growth between in vitro and in vivo, and therefore, the exploitation of hMSCs in new therapeutic strategies should be cautious under the malignant conditions.  2011.

title: Dkk-1 secreted by mesenchymal stem cells inhibits growth of breast cancer cells via depression of Wnt signalling.
abstract: We determined the molecular mechanism of inhibitory effect of human mesenchymal stem cells (hMSCs) on the growth of human MCF-7 breast cancer cells. Our finding showed that beta-catenin was down-regulated in MCF-7 cells by conditioned media from Z3 hMSCs, and the expression level of dickkopf-1 (Dkk-1) was higher in Z3 cells than that in MCF-7 cells. Neutralization of Dkk-1 and small interference RNA targeting Dkk-1 mRNA in Z3 cells attenuated the inhibitory effect of Z3 cells on MCF-7 cells. Overexpression of Dkk-1 in Z3 cells enhanced the inhibition. Therefore, Dkk-1 secreted by Z3 cells involves the inhibition via the Wnt pathway.

title: Cytosine deaminase‐producing human mesenchymal stem cells mediate an antitumor effect in a mouse xenograft model
abstract: Stem cell transplantation offers potential gene therapy for brain tumors. However, this approach has received little attention as a treatment for gastrointestinal tumors. In the present study, we explored the possibility of human bone marrow‐derived mesenchymal stem cells (hMSC) producing cytosine deaminase (CD), followed by systemic 5‐fluorocytosine (5‐FC) administration, showing an antitumor effect on a mouse gastric cancer xenograft.

title: Fibroblastic Colony‐Forming Unit Bone Marrow Cells Delay Progression to Gastric Dysplasia in a Helicobacter Model of Gastric Tumorigenesis
abstract: Bone marrow mesenchymal stem cells (MSCs) have been shown to have immune modulatory effects. Despite efforts to identify these cells in vivo, to date, MSCs have been defined mainly by their in vitro cell characteristics. Here, we show that Lin−CD44hiSca1−cKit+CD34− cells make up ∼0.5%–1% of murine whole bone marrow cells and yield nearly an equal amount of fibroblastic colony‐forming units (CFU‐F) as whole bone marrow. After transplantation into lethally irradiated recipients, Lin−CD44hiSca1−cKit+CD34− cells engrafted in the bone marrow long‐term and demonstrated characteristics of MSCs, including capacity to differentiate into osteoblasts and adipocytes. To examine whether Lin−CD44hiSca1−cKit+CD34− cells have immune modulatory effects, in vitro coculture with activated CD4+ T‐cells resulted in decreased Th17 cell differentiation by Lin−CD44hiSca1−cKit+CD34− cells. Furthermore, serial infusions with Lin−CD44hiSca1−cKit+CD34− cells reduced the progression to low‐grade gastric dysplasia in mice infected with chronic Helicobacter felis (p = .038). This correlated with reduced gastric interleukin (IL)‐17F, IL‐22, and ROR‐γt gene expression in responding mice (p < .05). These data suggest that bone marrow derived Lin−CD44hiSca1−cKit+CD34− cells have characteristics of MSCs and reduce progression of early gastric tumorigenesis induced by chronic H. felis infection. The prevention of dysplastic changes may occur through inhibition of Th17‐dependent pathways. STEM CELLS 2009;27:2301–2311

title: Mesenchymal stromal cells alone or expressing interferon-beta suppress pancreatic tumors in vivo, an effect countered by anti-inflammatory treatment.
abstract: Because of the inflammatory nature and extensive stromal compartment in pancreatic tumors, we investigated the role of mesenchymal stromal cells (MSC) to engraft selectively in pancreatic carcinomas and serve as anti-tumor drug delivery vehicles to control pancreatic cancer progression.



Human pancreatic carcinoma cells, PANC-1, expressing renilla luciferase were orthotopically implanted into SCID mice and allowed to develop for 10 days. Firefly luciferase-transduced MSC or MSC expressing interferon (IFN)-beta were then injected intraperitoneally weekly for 3 weeks. Mice were monitored by bioluminescent imaging for expression of renilla (PANC-1) and firefly (MSC) luciferase.



MSC selectively homed to sites of primary and metastatic pancreatic tumors and inhibited tumor growth (P=0.032). The production of IFN-beta within the tumor site by MSC-IFN-beta further suppressed tumor growth (P=0.0000083). Prior studies indicated that MSC home to sites of inflammation; therefore, we sought to alter the tumor microenvironment through treatment with a potent anti-inflammatory agent. After treatment, inflammation-associated mediators were effectively down-regulated, including NFkappaB, vascular endothelial growth factor (VEGF) and interleukin (IL)-6 as well as chemokines involved in MSC migration (CCL3 and CCL25). Treatment with the anti-inflammatory agent CDDO-Me before and after MSC-IFN-beta injections resulted in reduction of MSC in the tumors and reversed the positive effect of tumor inhibition by MSC-IFN-beta alone (P=0.041).



These results suggest that MSC exhibit innate anti-tumor effects against PANC-1 cells and can serve as delivery vehicles for IFN-beta for the treatment of pancreatic cancer. However, these beneficial effects may be lost in therapies combining MSC with anti-inflammatory agents.

title: Mesenchymal stem cells enhance growth and metastasis of colon cancer
abstract: Recently, mesenchymal stem cells (MSCs) were reported to migrate to tumor stroma as well as injured tissue. We examined the role of human MSCs in tumor stroma using an orthotopic nude mice model of KM12SM colon cancer. In in vivo experiments, systemically injected MSCs migrated to the stroma of orthotopic colon tumors and metastatic liver tumors. Orthotopic transplantation of KM12SM cells mixed with MSCs resulted in greater tumor weight than did transplantation of KM12SM cells alone. The survival rate was significantly lower in the mixed‐cell group, and liver metastasis was seen only in this group. Moreover, tumors resulting from transplantation of mixed cells had a significantly higher proliferating cell nuclear antigen labeling index, significantly greater microvessel area and significantly lower apoptotic index. Splenic injection of KM12SM cells mixed with MSCs, in comparison to splenic injection of KM12SM cells alone, resulted in a significantly greater number of liver metastases. MSCs incorporated into the stroma of primary and metastatic tumors expressed α‐smooth muscle actin and platelet‐derived growth factor receptor‐β as carcinoma‐associated fibroblast (CAF) markers. In in vitro experiments, KM12SM cells recruited MSCs, and MSCs stimulated migration and invasion of tumor cells through the release of soluble factors. Collectively, MSCs migrate and differentiate into CAFs in tumor stroma, and they promote growth and metastasis of colon cancer by enhancing angiogenesis, migration and invasion and by inhibiting apoptosis of tumor cells.

title: Prophylaxis against carcinogenesis in three kinds of unestablished tumor models via IL12-gene-engineered MSCs.
abstract: Mesenchymal stem cells (MSCs) were adenovirally engineered to secrete interleukin-12 (AdIL-12-MSCs) and evaluated for their anticarcinogenesis efficacy against three kinds of unestablished tumor models including B16 melanoma, LLC Lewis lung cancer and HCC hepatoma. Injection of AdIL-12-MSCs into protected mice before tumor inoculation prevented all of 12 mice in B16 preventive groups, 10 out of 12 in LLC lung cancer model and 11 out of 12 mice in HCC hepatoma model from developing tumors, whereas the control groups pre-receiving PBS were validated for 100% carcinogenesis; the tumor formation rates in free-AdIL-12 and vacant MSC groups were unveiled between approximately 83 and 100% even with plentiful angiogenesis and newborn lymphatic vessels, as well as distant metastases. As a novel approach, AdIL-12-MSC has revealed expected preventive effects on carcinogenesis (P<0.01) with low-toxic, broad-spectrum and long-range superiorities. In conclusion, our data indicate that AdIL-12-MSC possess the potential for tropism to preclinical tumor lesions and deprives surviving or hibernating tumor cells, which have escaped from conventional treatments, of revival and recurrence.

title: Mesenchymal stem cells within tumour stroma promote breast cancer metastasis
abstract: Mesenchymal stem cells have been recently described to localize to breast carcinomas, where they integrate into the tumour-associated stroma. However, the involvement of mesenchymal stem cells (or their derivatives) in tumour pathophysiology has not been addressed. Here, we demonstrate that bone-marrow-derived human mesenchymal stem cells, when mixed with otherwise weakly metastatic human breast carcinoma cells, cause the cancer cells to increase their metastatic potency greatly when this cell mixture is introduced into a subcutaneous site and allowed to form a tumour xenograft. The breast cancer cells stimulate de novo secretion of the chemokine CCL5 (also called RANTES) from mesenchymal stem cells, which then acts in a paracrine fashion on the cancer cells to enhance their motility, invasion and metastasis. This enhanced metastatic ability is reversible and is dependent on CCL5 signalling through the chemokine receptor CCR5. Collectively, these data demonstrate that the tumour microenvironment facilitates metastatic spread by eliciting reversible changes in the phenotype of cancer cells.