Paclitaxel-releasing mesenchymal stromal cells inhibit the growth of multiple myeloma cells in a dynamic 3D culture system.

Multiple myeloma is an aggressive tumour able to suppress osteoblastogenesis probably mediated by bone marrow mesenchymal stromal cells (BM-MSCs) that can also support plasma cell growth/survival. The use of MSCs for multiple myeloma therapy is a controversial topic because of the contradictory results on the capacity of MSCs to inhibit or to promote cancer growth. Our previous studies demonstrated that MSCs could be loaded with Paclitaxel (PTX) and used to deliver the drug in situ in amount affecting tumour growth (in vitro and in vivo). Therefore, independently on the discussed action of MSCs in myeloma, MSCs could represent a 'trojan horse' to vehicle and deliver anti-tumour agents into bone marrow. This study confirms, by an in vitro 3D dynamic culture system, that PTX loaded BM-MSCs (PTXr-MSCs) are active on the proliferation of RPMI 8226, a human myeloma cell line. Our results demonstrated a dramatic suppression of myeloma cell growth by PTXr-MSCs, suggesting that drug loaded MSCs could be a tool to deliver drug into the bone marrow. Drug releasing MSCs provide a therapeutic approach to potentiate the existing treatments against a very aggressive malignancy as multiple myeloma. Copyright © 2016 John Wiley & Sons, Ltd.

Gemcitabine-releasing mesenchymal stromal cells inhibit in vitro proliferation of human pancreatic carcinoma cells.

BACKGROUND AIMS: Pancreatic cancer (pCa) is a tumor characterized by a fibrotic state and associated with a poor prognosis. The observation that mesenchymal stromal cells (MSCs) migrate toward inflammatory micro-environments and engraft into tumor stroma after systemic administration suggested new therapeutic approaches with the use of engineered MSCs to deliver and produce anti-cancer molecules directly within the tumor. Previously, we demonstrated that without any genetic modifications, MSCs are able to deliver anti-cancer drugs. MSCs loaded with paclitaxel by exposure to high concentrations release the drug both in vitro and in vivo, inhibiting tumor proliferation. On the basis of these observations, we evaluated the ability of MSCs (from bone marrow and pancreas) to uptake and release gemcitabine (GCB), a drug widely used in pCa treatment. METHODS: MSCs were primed by 24-h exposure to 2000 ng/mL of GCB. The anti-tumor potential of primed MSCs was then investigated by in vitro anti-proliferation assays with the use of CFPAC-1, a pancreatic tumor cell line sensitive to GCB. The uptake/release ability was confirmed by means of high-performance liquid chromatography analysis. A cell-cycle study and secretome evaluation were also conducted to better understand the characteristics of primed MSCs. RESULTS: GCB-releasing MSCs inhibit the growth of a human pCa cell line in vitro. CONCLUSIONS: The use of MSCs as a "trojan horse" can open the way to a new pCa therapeutic approach; GCB-loaded MSCs that integrate into the tumor mass could deliver much higher concentrations of the drug in situ than can be achieved by intravenous injection.

Human CD14+ cells loaded with Paclitaxel inhibit in vitro cell proliferation of glioblastoma.

BACKGROUND AIMS: In attempting to develop new strategies to circumvent the immunosuppression associated with glioblastoma (GB), novel approaches have been designed using dendritic cell (DC)-based vaccination, which is considered a promising strategy to attack high-grade glioma. In previous studies, we demonstrated that human mesenchymal stromal cells without genetic manipulation but primed with Paclitaxel (PTX) acquire a potent anti-tumor activity, providing an interesting new biological approach for drug delivery. On the basis of these results, we here investigated whether both CD14+ and their derived DCs may behave like mesenchymal stromal cells acquiring anti-tumor activity on priming with PTX. METHODS: Human CD14+ cells were isolated from peripheral blood. Fluorescence-activated cell sorter analysis was performed to determine the purity of CD14+ and their differentiation into mature DCs. Cells were primed by incubation with 1 µg/mL of PTX for 24 h, and the PTX released by cells was assessed by mass spectrometry analysis. Anti-tumor activity was checked by testing the conditioned medium (CM) on the proliferation of U87 MG, a GB cell line. RESULTS: Both CD14+ and DCs were able to incorporate PTX and release the drug in the CM in a time-dependent manner (maximal release over 24 h). The addition of CM from CD14+ and DCs loaded with PTX strongly inhibits proliferation of U87 MG cells. CONCLUSIONS: Our results are the first demonstration that peripheral blood-derived CD14+ and DCs, in addition to their application for immunotherapy for GB, could also be used to delivery anti-cancer drugs, such as PTX, to kill GB cells.

Human mesenchymal stromal cells can uptake and release ciprofloxacin, acquiring in vitro anti-bacterial activity.

BACKGROUND AIMS: Traditional antibiotic therapy is based on the oral or systemic injection of antibiotics that are often unable to stop a deep infection (eg, osteomyelitis). We studied whether or not bone marrow stromal cells (BM-MSCs) are able to uptake and release ciprofloxacin (CPX), a fluoroquinolone considered the drug of choice for the treatment of chronic osteomyelitis because of its favorable penetration into poorly vascularized sites of infection. METHODS: Human bone marrow stromal cells (BM-MSCs) were primed with CPX (BM-MSCsCPX) according to a methodology previously standardized in our laboratory for paclitaxel (PTX). The anti-microbial activity of CPX released from BM-MSCs cells (BM-MSCsCPX-CM) or supernatant from cell lysate (BM-MSCsCPX-LYS) was evaluated by agar dilution and microdilution methods on three bacterial strains (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa). To investigate whether or not primed cells (BM-MSCsCPX) were able to directly act on the bacterial growth, co-colture was performed by mixing E. coli suspension to an increasing number of BM-MSCsCPX. The anti-bacterial activity was determined as number of BM-MSCsCPX that completely inhibited bacterial growth. RESULTS: The results demonstrated that BM-MSCsCPX are able to uptake and then release CPX in the conditioned medium. The loaded antibiotic maintains its active form throughout the process as tested on bacteria. CONCLUSIONS: Our findings suggest that CPX-loaded MSCs may represent an important device for carrying and delivering CPX (and perhaps other antibiotics) into infected deep microenvironments; they could be used for local application and by systemic infusion when their homing capacity into the bone is cleared.

Mesenchymal stromal cells primed with Paclitaxel attract and kill leukaemia cells, inhibit angiogenesis and improve survival of leukaemia-bearing mice.

Current leukaemia therapy focuses on increasing chemotherapy efficacy. Mesenchymal stromal cells (MSCs) have been proposed for carrying and delivery drugs to improve killing of cancer cells. We have shown that MSCs loaded with Paclitaxel (PTX) acquire a potent anti-tumour activity. We investigated the effect of human MSCs (hMSCs) and mouse SR4987 loaded with PTX (hMSCsPTX and SR4987PTX) on MOLT-4 and L1210, two leukaemia cell (LCs) lines of human and mouse origin, respectively. SR4987PTX and hMSCsPTX showed strong anti-LC activity. hMSCsPTX, co-injected with MOLT-4 cells or intra-tumour injected into established subcutaneous MOLT-4 nodules, strongly inhibited growth and angiogenesis. In BDF1-mice-bearing L1210, the intraperitoneal administration of SR4987PTX doubled mouse survival time. In vitro, both hMSCs and hMSCsPTX released chemotactic factors, bound and formed rosettes with LCs. In ultrastructural analysis of rosettes, hMSCsPTX showed no morphological alterations while the attached LCs were apoptotic and necrotic. hMSCs and hMSCsPTX released molecules that reduced LC adhesion to microvascular endothelium (hMECs) and down-modulated ICAM1 and VCAM1 on hMECs. Priming hMSCs with PTX is a simple procedure that does not require any genetic cell manipulation. Once the effectiveness of hMSCsPTX on established cancers in mice is proven, this procedure could be proposed for leukaemia therapy in humans.

A mesenchymal stromal cell line resistant to paclitaxel that spontaneously differentiates into osteoblast-like cells.

The mesenchymal stromal cell line SR-4987 has been established in our laboratory from the bone marrow of BDF/1 mice. Recent information on mesenchymal stem cells biology and the need to deal with well-characterized cell lines suggest to critically consider the existent data on this cell line by updating them with new investigations on growth parameters, in vitro plasticity, and drug sensitivity to anti-cancer, anti-inflammatory, and a histone deacetylase inhibitor. SR-4987 cells show a population doubling time of 24.5 ± 5.4 h, a plating efficiency of 2.87 ± 1.19%, and under stimulation maintain only in part their multipotency by differentiating towards chondro-osteogenic lineages but not into adipogenic. Surprisingly, these mesenchymal stromal cells differentiate spontaneously into osteoblast-like cells and this is significantly stimulated by valproic acid. SR-4987 cells show a dramatic resistance to paclitaxel (PTX) with a resistance index of 39.6 times (evaluated versus MOLT-4 leukemia) and of 68.2 (versus HT-29 colorectal carcinoma). SR-4987 resistance is reversed by verapamil and correlates with high expression of P-glycoprotein that is down-modulated by PTX. Taken together, our results indicated that SR-4987 line is a very interesting cell model useful to investigate both drug sensitivity resistance and physiopathological aspects related to mesenchymal cell function.

Molecular analysis of relapses or reinfections of Clostridium difficile-associated diarrhea.

Recurrence is a major complication of Clostridium difficile-associated diarrhea and occurs in 15 to 20% of patients after discontinuation of therapy. Strains from 53 patients with Clostridium difficile recurrences were fingerprinted by PCR ribotyping. Reinfection with a different strain occurred in 15 out of 53 patients (28,3%), while 38 patients relapsed. These data suggest the need to perform molecular typing for implementation of infection control procedures and for a more appropriate therapeutic strategy.

CD45+/CD133+ positive cells expanded from umbilical cord blood expressing PDX-1 and markers of pluripotency.

UCB (human umbilical cord blood) contains cells able to differentiate into non-haematopoietic cell lineages. It also contains cells similar to primitive ESCs (embryonic stem cells) that can differentiate into pancreatic-like cells. However, few data have been reported regarding the possibility of expanding these cells or the differential gene expression occurring in vitro. In this study, we expanded formerly frozen UCB cells by treatment with SCF (stem cell factor) and GM-CSF (granulocyte-macrophage colony stimulating factor) in the presence of VPA (valproic acid). Gene expression profiles for beta cell differentiation and pluripotency (embryo stem cell phenotype) were analysed by RT-PCR and immunocytochemistry. The results show a dramatic expansion (>150-fold) of haematopoietic progenitors (CD45+/CD133+) which also expressed embryo markers of pluripotency (nanog, kfl-4, sox-2, oct-3/4 and c-myc), nestin, and pancreatic markers such as pax-4, ngn-3, pdx-1 and syt-1 (that is regulated by pdx-1 and provides the cells with a Ca++ regulation mechanism essential for insulin exocytosis). Our results show that UCB cells can be expanded to produce large numbers of cells of haematopoietic lineage that naturally (without the need of retroviral vectors or transposons) express a gene pattern compatible with endocrine pancreatic precursors and markers of pluripotency. Further investigations are necessary to clarify, first, whether in this context, the embryogenes expressed are functional or not, and secondly, since these cells are safer than cells transfected with retroviral vectors or transposons, whether they would represent a potential tool for clinical application.

Prevalidation of the rat CFU-GM assay for in vitro toxicology applications.

In vitro haematotoxicity assays are thought to have the potential to significantly reduce and refine the use of animals for haematotoxicity testing. These assays are used successfully in all types of studies - however, their use is not so common in human toxicology studies in the preclinical setting, as they are not required for regulatory testing in this case. Furthermore, these assays could play a key role in bridging the gap between preclinical toxicology studies in animal models and clinical investigations. In previous studies, the Colony Forming Unit-Granulocyte Macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood) and for predicting the in vivo human maximal tolerated dose (MTD) by adjusting in vivo data on mouse toxicity. Recently, a Colony Forming Unit-Megakaryocyte (CFU-MK) assay has also been prevalidated for testing drug toxicity toward megakaryocytes. The rat CFU-GM assay has been used by many researchers for its ability to evaluate in vitro haematotoxicity. Although it is not yet available, a standardised procedure for data comparison could be very important, since the rat is the most widely-used species for the in vivo testing of toxicants. This report presents the results of the prevalidation study developed to analyse the intra-laboratory and inter-laboratory variability of a standardised operating procedure for this assay and its performance for the in vitro determination of the inhibitory concentration (IC) values of drugs on rat myeloid progenitors (CFU-GM). The results demonstrate that the CFU-GM assay can be performed with cryopreserved rat bone-marrow cells (rBMC). The assay represents a useful tool for evaluating the toxicity of a compound, in terms of both relative toxicity (when different molecules are compared) and the prediction of the degree of in vivo toxicity. The use of this assay could greatly reduce the number of rats used in experimental procedures, and could also contribute to the accumulation of more toxicity data on compounds to be registered according to the criteria established by the European Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) programme.

Paclitaxel Priming of TRAIL Expressing Mesenchymal Stromal Cells (MSCs-TRAIL) Increases Antitumor Efficacy of Their Secretome.

BACKGROUND: Adipose tissue derived MSCs engineered with the tumor necrosis factor-related apoptosis-inducing ligand protein (MSCs-TRAIL) have a significant anticancer activity. MSCs, without any genetic modifications, exposed to high doses of chemotherapeutic agents are able to uptake the drug and release it in amount affecting tumor proliferation. The purpose of this study was to verify the ability of MSCs-TRAIL to uptake and release paclitaxel (PTX) by providing an increased antitumor efficacy. METHODS: MSCs and MSCs-TRAIL were tested for their sensitivity to Paclitaxel (PTX) by MTT assay and the cells were loaded with PTX according to a standardized procedure. The secretome was analysed by HPLC for the presence of PTX, microarray assay for soluble TRAIL (s-TRAIL) and tested for in vitro anticancer activity. RESULTS: MSCs-TRAIL were resistant to PTX and able to incorporate and then release the drug. The secretion of s-TRAIL by PTX loaded MSCs-TRAIL was not inhibited and the PTX delivery together with s-TRAIL secretion resulted into an increased antitumor efficacy of cell secretoma as tested in vitro on human pancreatic carcinoma (CFPAC-1) and glioblastoma (U87-MG). CONCLUSIONS: Our result is the first demonstration of the possible merging of two new MSCs therapy approaches based on genetic manipulation and drug delivery. If confirmed in vivo, this could potentiate the efficacy of MSCs-TRAIL and strongly contribute to reduce the toxicity due to the systemic treatment of PTX.

Antimicrobial susceptibility testing of Helicobacter pylori determined by microdilution method using a new medium.

Antibiotic susceptibility testing of Helicobacter pylori isolates was performed by broth microdilution method with MegaCell RPMI-1640 Medium (SIGMA). Fifty five clinical isolates of H. pylori were tested against metronidazole, tinidazole, amoxicillin, and clarithromycin. The results were compared to those obtained by standard agar dilution method. The microdilution method performed with new medium, showed excellent correlation with agar dilution results, with 100% agreement for metronidazole, 96.3% for amoxicillin, 90.7% for clarithromycin, and 92.8% for tinidazole. MICs determined by proposed method were highly reproducible: replicate results were variable within one-two-fold dilution by using different inocula and different batches of medium.

Refinement and optimisation of the rat CFU-GM assay to incorporate the use of cryopreserved bone-marrow cells for in vitro toxicology applications.

The colony-forming unit-granulocyte-macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood cells) and for predicting in vivo human Maximal Tolerated Dose (MTD) values by extrapolating in vivo data on mouse toxicity. The rat CFU-GM assay is widely used for its capability to evaluate in vitro haematotoxicity, but no standardised procedure suitable for data comparison has been developed. A validated rat CFU-GM assay is needed for many reasons - not least because the rat is the most commonly-used species for the in vivo testing of toxicants. This report describes the refinement and optimisation of a standardised protocol for entering into the prevalidation phase of test development. The sensitivity of rat progenitors to granulocyte-macrophage-colony stimulating factor (GM-CSF), the correlation between the number of cells seeded and the number of colonies obtained, the role of mesenchymal cells on CFU-GM proliferation and the performance of the assay, and the effects of using different types of plastic dishes and sources of cytokines, are described. A standard operating procedure (SOP) based on the use of cryopreserved progenitors has been generated, to be applied to the in vitro toxicity testing of compounds. This SOP dramatically reduces the number of rats used and increases the homogeneity of the data obtained.

Human mesenchymal stromal cells inhibit tumor growth in orthotopic glioblastoma xenografts.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) represent an attractive tool for cell-based cancer therapy mainly because of their ability to migrate to tumors and to release bioactive molecules. However, the impact of MSCs on tumor growth has not been fully established. We previously demonstrated that murine MSCs show a strong tropism towards glioblastoma (GBM) brain xenografts and that these cells are able to uptake and release the chemotherapeutic drug paclitaxel (PTX), maintaining their tropism towards the tumor. Here, we address the therapy-relevant issue of using MSCs from human donors (hMSCs) for local or systemic administration in orthotopic GBM models, including xenografts of patient-derived glioma stem cells (GSCs). METHODS: U87MG or GSC1 cells expressing the green fluorescent protein (GFP) were grafted onto the striatum of immunosuppressed rats. Adipose hMSCs (Ad-hMSCs), fluorescently labeled with the mCherry protein, were inoculated adjacent to or into the tumor. In rats bearing U87MG xenografts, systemic injections of Ad-hMSCs or bone marrow (BM)-hMSCs were done via the femoral vein or carotid artery. In each experiment, either PTX-loaded or unloaded hMSCs were used. To characterize the effects of hMSCs on tumor growth, we analyzed survival, tumor volume, tumor cell proliferation, and microvascular density. RESULTS: Overall, the AD-hMSCs showed remarkable tropism towards the tumor. Intracerebral injection of Ad-hMSCs significantly improved the survival of rats with U87MG xenografts. This effect was associated with a reduction in tumor growth, tumor cell proliferation, and microvascular density. In GSC1 xenografts, intratumoral injection of Ad-hMSCs depleted the tumor cell population and induced migration of resident microglial cells. Overall, PTX loading did not significantly enhance the antitumor potential of hMSCs. Systemically injected Ad- and BM-hMSCs homed to tumor xenografts. The efficiency of hMSC homing ranged between 0.02 and 0.5% of the injected cells, depending both on the route of cell injection and on the source from which the hMSCs were derived. Importantly, systemically injected PTX-loaded hMSCs that homed to the xenograft induced cytotoxic damage to the surrounding tumor cells. CONCLUSIONS: hMSCs have a therapeutic potential in GBM brain xenografts which is also expressed against the GSC population. In this context, PTX loading of hMSCs seems to play a minor role.

Bcl-2 down modulation in WEHI-3B/CTRES cells resistant to Cholera Toxin (CT)-induced apoptosis.

The very different effects of Cholera Toxin (CT) on cell growth and proliferation may depend on the type of ganglioside receptors in cell membranes and different signal transduction mechanisms triggered, but other functions related to the drug resistance mechanisms can not be excluded. The effect of CT treatment on the "in vitro" clonogenicity, the Population Doubling Time (PDT), apoptosis, PKA activation and Bax and Bcl-2 expression was evaluated in WEHI-3B cell line and its CT-resistant subclone (WEHI-3B/CTRES). In WEHI-3B parental cells the dramatic accumulation of cAMP induced by CT correlated well with PKA activation, increased PDT value, inhibition of clonogenicity and apoptosis. H-89 treatment inhibited PKA activation by CT but did not protect the cells from apoptosis and growth inhibition. In WEHI-3B/CTRES no significant CT-dependent accumulation of cAMP occurred with any increase of PKA activity and PDT. In CT resistant cells (WEHI-3B/CTRES), Bcl-2 expression was down regulated by both CT or drug treatment (eg., ciprofloxacin, CPX) although these cells were protected from CT-dependent apoptosis but not from drug-induced apoptosis. Differently from other cell models described, down regulation of Bcl-2 is proved to be independent on cAMP accumulation and PKA activation. Our observations support the implication of cAMP dependent kinase (PKA) in the inhibition of WEHI-3B cells growth and suggest that, in WEHI-3B/CTRES, Bcl-2 expression could be modulated by CT in the absence of cAMP accumulation. Also in consideration of many contradictory data reported in literature, our cell models (of one sensitive parental cell strain and two clones with different uncrossed specific resistance to CT and CPX) provides a new and interesting tool for better investigating the relationship between the CT signal transduction mechanisms and Bcl-2 expression and function.

In vitro activity of artemisone and artemisinin derivatives against extracellular and intracellular Helicobacter pylori.

The in vitro activity of the new artemisinin derivative artemisone as well as other molecules of the same class against Helicobacter pylori and their effects when combined with standard antibiotics were evaluated. Since H. pylori can be internalised into gastric epithelial cells, the effects of artemisinin, dihydroartemisinin and artemisone against intracellular H. pylori were also investigated. Bacteriostatic [minimum inhibitory concentration (MIC)] and bactericidal [minimum bactericidal concentration (MBC)] activities were assessed against 24 clinical strains of H. pylori with different antibiotics susceptibilities. Artemisone showed MIC50 and MIC90 values of 0.25 mg/L and 0.5 mg/L, respectively, and an MBC50 value of 0.5 mg/L. Artemisone was synergistic with amoxicillin in 60% of strains, with clarithromycin in 40% and with metronidazole in 20%. There was no interaction between artemisone and omeprazole or bismuth citrate. Against intracellular H. pylori, only dihydroartemisinin at 2× MIC caused a 1 log10 CFU decrease after 18 h and 24 h of incubation. This is the first demonstration in vitro of the activity of artemisinin derivatives against intracellular H. pylori and indicates that artemisone has the potential to be efficacious for the treatment of H. pylori infection, especially in combination with antibiotics.

Angiogenic and anti-inflammatory properties of micro-fragmented fat tissue and its derived mesenchymal stromal cells.

BACKGROUND: Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for advanced cell-based therapies. They are routinely obtained enzymatically from fat lipoaspirate (LP) as SVF, and may undergo prolonged ex vivo expansion, with significant senescence and decline in multipotency. Besides, these techniques have complex regulatory issues, thus incurring in the compelling requirements of GMP guidelines. Hence, availability of a minimally manipulated, autologous adipose tissue would have remarkable biomedical and clinical relevance. For this reason, a new device, named Lipogems® (LG), has been developed. This ready-to-use adipose tissue cell derivate has been shown to have in vivo efficacy upon transplantation for ischemic and inflammatory diseases. To broaden our knowledge, we here investigated the angiogenic and anti-inflammatory properties of LG and its derived MSC (LG-MSCs) population. METHODS: Human LG samples and their LG-MSCs were analyzed by immunohistochemistry for pericyte, endothelial and mesenchymal stromal cell marker expression. Angiogenesis was investigated testing the conditioned media (CM) of LG (LG-CM) and LG-MSCs (LG-MSCs-CM) on cultured endothelial cells (HUVECs), evaluating proliferation, cord formation, and the expression of the adhesion molecules (AM) VCAM-1 and ICAM-1. The macrophage cell line U937 was used to evaluate the anti-inflammatory properties, such as migration, adhesion on HUVECs, and release of RANTES and MCP-1. RESULTS: Our results indicate that LG contained a very high number of mesenchymal cells expressing NG2 and CD146 (both pericyte markers) together with an abundant microvascular endothelial cell (mEC) population. Substantially, both LG-CM and LG-MSC-CM increased cord formation, inhibited endothelial ICAM-1 and VCAM-1 expression following TNFα stimulation, and slightly improved HUVEC proliferation. The addition of LG-CM and LG-MSC-CM strongly inhibited U937 migration upon stimulation with the chemokine MCP-1, reduced their adhesion on HUVECs and significantly suppressed the release of RANTES and MCP-1. CONCLUSIONS: Our data indicate that LG micro-fragmented adipose tissue retains either per se, or in its embedded MSCs content, the capacity to induce vascular stabilization and to inhibit several macrophage functions involved in inflammation.

Cell-mediated drug delivery by gingival interdental papilla mesenchymal stromal cells (GinPa-MSCs) loaded with paclitaxel.

OBJECTIVE: Gingival tissue is composed of cell types that contribute to the body's defense against many agents in oral environment, wound healing and tissue regeneration. Thanks to their easy and scarcely invasive withdrawal procedure, interdental papilla provide a good source of mesenchymal stromal cells (GinPa-MSCs). We isolated GinPa-MSCs and verified their ability to uptake/release the anticancer agent Paclitaxel (PTX). METHODS: In vitro expanded GinPa-MSCs were characterized for CD markers by FACS, tested for differentiation ability and analyzed by TEM. Their ability to uptake/release PTX was assessed according to a standardized procedure. RESULTS: The CD expression and chondro-adipo-osteo differentiation ability confirmed the mesenchymal feature of GinPa-MSCs. Surprisingly, 28% of GinPa-MSCs expressed CD14 marker and had an impressive pinocytotic activity. GinPa-MSCs were able to take up and release a sufficient amount of PTX to demonstrate effective in vitro activity against pancreatic carcinoma cells, suggesting that the drug was not inactivated. CONCLUSIONS: The procedure to obtain MSCs from interdental papilla is less invasive than that used for both bone marrow and adipose tissue, GinPa-MSCs are easy to expand and can be efficiently loaded with PTX. Taken together these qualities suggest that GinPa-MSCs may prove to be a good tool for cell-mediated drug delivery in cancer, particularly if related to stomatognathic system.

Human amniotic mesenchymal stromal cells (hAMSCs) as potential vehicles for drug delivery in cancer therapy: an in vitro study.

INTRODUCTION: In the context of drug delivery, mesenchymal stromal cells (MSCs) from bone marrow and adipose tissue have emerged as interesting candidates due to their homing abilities and capacity to carry toxic loads, while at the same time being highly resistant to the toxic effects. Amongst the many sources of MSCs which have been identified, the human term placenta has attracted particular interest due to its unique, tissue-related characteristics, including its high cell yield and virtually absent expression of human leukocyte antigens and co-stimulatory molecules. Under basal, non-stimulatory conditions, placental MSCs also possess basic characteristics common to MSCs from other sources. These include the ability to secrete factors which promote cell growth and tissue repair, as well as immunomodulatory properties. The aim of this study was to investigate MSCs isolated from the amniotic membrane of human term placenta (hAMSCs) as candidates for drug delivery in vitro. METHODS: We primed hAMSCs from seven different donors with paclitaxel (PTX) and investigated their ability to resist the cytotoxic effects of PTX, to upload the drug, and to release it over time. We then analyzed whether the uptake and release of PTX was sufficient to inhibit proliferation of CFPAC-1, a pancreatic tumor cell line sensitive to PTX. RESULTS: For the first time, our study shows that hAMSCs are highly resistant to PTX and are not only able to uptake the drug, but also release it over time. Moreover, we show that PTX is released from hAMSCs in a sufficient amount to inhibit tumor cell proliferation, whilst some of the PTX is also retained within the cells. CONCLUSION: Taken together, for the first time our results show that placental stem cells can be used as vehicles for the delivery of cytotoxic agents.

Mesenchymal stromal cells uptake and release paclitaxel without reducing its anticancer activity.

To improve the drug delivery efficiency on target cells, many strategies have been developed including Mesenchymal Stromal Cells (MSCs) approaches. In a previous study, we found that bone-marrow-derived MSCs (BM-MSCs) were able to incorporate and release the anti-tumor and anti-angiogenic drug, Paclitaxel (PTX). In this study, we evaluated the stability of PTX in standard cell culture conditions by analyzing the metabolites produced by MSCs after their incorporation of the drug. We are able to show that MSCs do not release either 3-OH-PTX or 6-OH-PTX metabolites (having a lower anticancer activity) but release an active PTX molecule together with the isomer 7-Epitaxol, is known to maintain the whole biological activity. This confirms that the simple procedure of MSCs priming with a drug (without any genetic cell manipulation), in our case PTX, does not modify the activity of the molecule and provides a new biological-device to carry and deliver PTX in tumor sites, by contributing to improve drug efficacy and target selectivity in cancer therapy.

Mesenchymal stromal cells loaded with paclitaxel induce cytotoxic damage in glioblastoma brain xenografts.

INTRODUCTION: The goal of cancer chemotherapy is targeting tumor cells and/or tumor-associated microvessels with the lowest systemic toxicity. Mesenchymal stromal cells (MSCs) are promising vehicles for selective drug delivery due to their peculiar ability to home to pathological tissues. We previously showed that MSCs are able to uptake and subsequently to release the chemotherapeutic compound Paclitaxel (PTX) and to impair the growth of subcutaneous glioblastoma multiforme (GBM) xenografts. Here we used an orthotopic GBM model 1) to assess whether PTX-loaded MSCs (PTX-MSCs) retain a tropism towards the tumor cells in the brain context, and 2) to characterize the cytotoxic damage induced by MSCs-driven PTX release in the tumor microenvironment. METHODS: U87MG GBM cells were fluorescently labeled with the mCherry protein and grafted onto the brain of immunosuppressed rats. In adjacent brain regions, we injected green fluorescent protein-expressing murine MSCs, either loaded with PTX or unloaded. After 1 week survival, the xenografted brain was assessed by confocal microscopy for PTX-induced cell damage. RESULTS: Overall, MSCs showed remarkable tropism towards the tumor. In rats grafted with PTX-MSCs, the nuclei of U87MG cells showed changes that are typically induced by PTX, including multi-spindle mitoses, centrosome number alterations, and nuclear fragmentation. Multi-spindle mitoses resulted in multinucleated cells that were significantly higher in tumors co-grafted with PTX-MSCs than in controls. Nuclear changes did not occur in astrocytes and neurons surrounding the tumor. CONCLUSIONS: MSCs appear particularly suited for anti-neoplastic drug delivery in the brain since PTX-specific damage of GBM cells can be achieved avoiding side effects to the normal tissue.