RESUMEN
Triple-negative breast cancer (TNBC) is a very aggressive disease even in its early stages and is characterized by a severe prognosis. Neoadjuvant chemotherapy is one of the milestones of treatment, and paclitaxel (PTX) is among the most active drugs used in this setting. However, despite its efficacy, peripheral neuropathy occurs in approximately 20-25% of cases and represents the dose-limiting toxicity of this drug. New deliverable strategies to ameliorate drug delivery and reduce side effects are keenly awaited to improve patients' outcomes. Mesenchymal stromal cells (MSCs) have recently been demonstrated as promising drug delivery vectors for cancer treatment. The aim of the present preclinical study is to explore the possibility of a cell therapy approach based on the use of MSCs loaded with PTX to treat TNBC-affected patients. For this purpose, we in vitro evaluated the viability, migration and colony formation of two TNBC cell lines, namely, MDA-MB-231 and BT549, treated with MSC-PTX conditioned medium (MSC-CM PTX) in comparison with both CM of MSCs not loaded with PTX (CTRL) and free PTX. We observed stronger inhibitory effects on survival, migration and tumorigenicity for MSC-CM PTX than for CTRL and free PTX in TNBC cell lines. Further studies will provide more information about activity and potentially open the possibility of using this new drug delivery vector in the context of a clinical study.
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Células Madre Mesenquimatosas , Neoplasias de la Mama Triple Negativas , Humanos , Paclitaxel/uso terapéutico , Neoplasias de la Mama Triple Negativas/metabolismo , Medios de Cultivo Condicionados/farmacología , Medios de Cultivo Condicionados/metabolismo , Línea Celular Tumoral , Células Madre Mesenquimatosas/metabolismoRESUMEN
BACKGROUND: In vitro primary cultures of microvascular endothelial cells from endocrine pancreas are difficult to obtain, but can be a very helpful tool for studies of islet biology, transplantation and regenerative medicine. METHODS: We applied a protocol recently described for the isolation and culture of brain microvascular endothelial cells (EC) on human pancreatic islets. EC obtained were characterized in terms of morphological (light and transmission electron microscopy), phenotypical (by immunofluorescence and flow cytometry) and functional (cord formation assay and protein secretion by multiplex bead-based assay) characteristics. RESULTS: EC were obtained from 25% of islet preparations processed. Two primary endothelial cell lines showed high proliferative potential and were deeply characterized: they presented endothelial cell morphology and expressed CD31, CD49a, CD49e, CD34, von Willebrand Factor (vWF), Vascular Endothelial CAdherin (VE-CAD), Tyrosine Kinase with Ig and EGF Homology Domains-2 (TIE2), Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), Ulex lectin and the endothelium endocrine-specific marker nephrin. Besides, they were able to form cordons in vitro and secreted factors involved in the process of angiogenesis such as Vascular Endothelial Growth Factor (VEGF), Monocyte Chemotactic Protein 1 (MCP-1), interleukin (IL)-8 and Melanoma Growth Stimulatory Activity Alpha (GROα). These cell lines were termed Human Islet Microvascular Endothelial Cells (HIMEC). DISCUSSION: This study establishes a simple and effective strategy for isolation and long-term culture of EC derived from human pancreatic islet. HIMEC in culture preserve phenotype and functional properties and are, therefore, a useful tool for future experiments of in vitro pancreas modelling, co-transplantation with pancreatic islets, re-vascularization of scaffold or matrix for regenerative medicine purposes.
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Células Endoteliales/metabolismo , Endotelio Vascular/citología , Islotes Pancreáticos/citología , Antígenos CD/metabolismo , Cadherinas/metabolismo , Células Cultivadas , Células Endoteliales/citología , Humanos , Interleucina-8/metabolismo , Microvasos/citología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: Glioblastoma (GBM) represents the most aggressive malignant brain tumor in adults, with a risible median life expectancy despite gold standard treatment. Novel drug-delivery methods have been explored. Here we evaluated the possibility to use mononuclear cells (MCs) belonging to the monocytic-dendritic lineage as drug-carrier. METHODS: MCs were obtained from 10 patients harboring a GBM, and from healthy volunteers, considered as controls. GBM tissue was also obtained from patients. MCs were cultured and the adherent population on fibronectin (FN-MCs), after immunocytochemistry and flow cytometry characterization, was loaded with Paclitaxel (FN-MCs-PTX). Antiproliferative and migration activity of FN-MCs-PTX was evaluated in two-dimensional (2D) and three-dimensional (3D) co-culture assays with red fluorescent U87 Malignant Glioma cells and primary GBM cells. Antiangiogenic properties of FN-MCs-PTX were tested on cultures with endothelial cells. RESULTS: Phenotypical characterization showed a high expression of monocytic-dendritic markers in GBM cells and FN-MCs. FN-MCs demonstrated to effectively uptake PTX and to strongly inhibit GBM growth in vitro (P <0.01). Moreover, tumor-induced migration of MCs, although partially affected by the PTX cargo, remained statistically significant when compared with unprimed cells and this was confirmed in a 3D Matrigel model (P <0.01) and in a Trans-well assay (P <0.01). FN-MCs-PTX also disclosed considerable antiangiogenic properties. DISCUSSION: Our results suggest that the fibronectin-adherent population of MCs isolated from peripheral blood can be an effective tool to inhibit GBM growth. Given the relative facility to obtain such cells and the short time needed for their culture and drug loading this approach may have potential as an adjuvant therapy for GBM.
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Antineoplásicos Fitogénicos/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Fibronectinas/metabolismo , Glioblastoma/tratamiento farmacológico , Paclitaxel/administración & dosificación , Adulto , Anciano , Adhesión Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Portadores de Fármacos , Humanos , Leucocitos Mononucleares/citología , Persona de Mediana EdadRESUMEN
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.
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Antineoplásicos/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Mieloma Múltiple/metabolismo , Paclitaxel/farmacología , Línea Celular Tumoral , Proliferación Celular , Medios de Cultivo Condicionados , Resistencia a Antineoplásicos , Tolerancia a Medicamentos , Humanos , Mieloma Múltiple/patología , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
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.
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Antineoplásicos/administración & dosificación , Medios de Cultivo Condicionados/farmacología , Células Dendríticas/inmunología , Glioblastoma/terapia , Células Madre Mesenquimatosas , Paclitaxel/administración & dosificación , Vacunas contra el Cáncer/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Sistemas de Liberación de Medicamentos , Glioblastoma/inmunología , Glioblastoma/patología , Humanos , Receptores de Lipopolisacáridos/análisis , Trasplante de Células Madre MesenquimatosasRESUMEN
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.
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Antineoplásicos/administración & dosificación , Desoxicitidina/análogos & derivados , Sistemas de Liberación de Medicamentos/métodos , Células Madre Mesenquimatosas/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Desoxicitidina/administración & dosificación , Humanos , Paclitaxel/administración & dosificación , Gemcitabina , Neoplasias PancreáticasRESUMEN
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.
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Antibacterianos/uso terapéutico , Huesos/efectos de los fármacos , Huesos/patología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Ciprofloxacina/uso terapéutico , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Osteomielitis/terapia , Antibacterianos/metabolismo , Actividad Bactericida de la Sangre/efectos de los fármacos , Células Cultivadas , Enfermedad Crónica , Ciprofloxacina/metabolismo , Endocitosis , Exocitosis , HumanosRESUMEN
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.
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Comunicación Celular/fisiología , Leucemia/patología , Leucemia/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/patología , Paclitaxel/farmacología , Animales , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Leucemia/tratamiento farmacológico , Leucemia/cirugía , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Desnudos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Malignant pleural mesothelioma is an asbestos-related tumor originating in mesothelial cells of the pleura that poorly responds to chemotherapeutic approaches. Adult mesenchymal stromal cells derived either from bone marrow or from adipose tissue may be considered a good model for cell-based therapy, a treatment which has experienced significant interest in recent years. The present study confirms that Paclitaxel is effective on mesothelioma cell proliferation in 2D and 3D in vitro cultures, and that 80,000 mesenchymal stromal cells loaded with Paclitaxel inhibit tumor growth at a higher extent than Paclitaxel alone. An in vivo approach to treat in situ mesothelioma xenografts using a minimal amount of 106 mesenchymal stromal cells loaded with Paclitaxel showed the same efficacy of a systemic administration of 10 mg/kg of Paclitaxel. These data strongly support drug delivery system by mesenchymal stromal cells as a useful approach against many solid tumors. We look with interest at the favourable opinion recently expressed by the Italian Drug Agency on the procedure for the preparation of mesenchymal stromal cells loaded with Paclitaxel in large-scale bioreactor systems and their storage until clinical use. This new Advanced Medicinal Therapy Product, already approved for a Phase I clinical trial on mesothelioma patients, could pave the way for mesenchymal stromal cells use as drug delivery system on other solid tumors for adjuvant therapy associated with surgery and radiotherapy.
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Células Madre Mesenquimatosas , Mesotelioma Maligno , Mesotelioma , Humanos , Paclitaxel , Línea Celular Tumoral , Mesotelioma/tratamiento farmacológicoRESUMEN
BACKGROUND: Osteosarcoma (OS) represents a rare cancer with an unfavorable prognosis that needs innovative treatment. The aim was to isolate a secretome from mesenchymal stem cells (MSCs) that are treated with paclitaxel (PTX)-containing microvesicles as a drug delivery system and analyze its cytotoxic effects on OS cell lines (SJSA, MG63, and HOS). METHODS: Three batches of secretome (SECR-1, SECR-2, and SECR-3) were produced from three bone marrow (BM) MSCs samples treated for 24 h with 15 µg/mL of PTX or with a standard medium. The viability of the OS cell lines after 5 days of exposure to SECR-1-2-3 (pure and diluted to 1:2 and 1:4) was analyzed with an MTT assay. The same SECR batches were analyzed with high-performance liquid chromatography (HPLC) and with a nanoparticle tracking assay (NTA). RESULTS: A statistically significant decrease in the viability of all OS cell lines was observed after treatment with SECR-PTX 1-2-3 in a dose-response manner. The NTA analyses showed the presence of nanoparticles (NPs) with a mean size comparable to that of extracellular vesicles (EVs). The HPLC analyses detected the presence of PTX in minimal doses in all SECR batches. CONCLUSIONS: This proof-of-concept study showed that the conditioned medium isolated from MSCs loaded with PTX had a strong cytotoxic effect on OS cell lines, due to the presence of EV and PTX.
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BACKGROUND: Malignant pleural mesothelioma is a pathology with no effective therapy and a poor prognosis. Our previous study demonstrated an in vitro inhibitory effect on mesothelioma cell lines of both the lysate and secretome of adipose tissue-derived Mesenchymal Stromal Cells. The inhibitory activity on tumor growth has been demonstrated also in vivo: five million Mesenchymal Stromal Cells, injected "in situ", produced a significant therapeutic efficacy against MSTO-211H xenograft equivalent to that observed after the systemic administration of paclitaxel. OBJECTIVE: The objective of this study is to evaluate the efficacy of low amount (half a million) Mesenchymal Stromal Cells and micro-fragmented adipose tissues (the biological tissue from which the Mesenchymal Stromal Cells were isolated) on mesothelioma cells growth. METHODS: Tumor cells growth inhibition was evaluated in vitro and in a xenograft model of mesothelioma. RESULTS: The inhibitory effect of micro-fragmented fat from adipose-tissue has been firstly confirmed in vitro on MSTO-211H cell growth. Then the efficacy against the growth of mesothelioma xenografts in mice of both micro-fragmented fat and low amount of Mesenchymal Stromal Cells has been evaluated. Our results confirmed that both Mesenchymal Stromal Cells and micro-fragmented fat, injected "in situ", did not stimulate mesothelioma cell growth. By contrast, micro-fragmented fat produced a significant inhibition of tumor growth and progression, comparable to that observed by the treatment with paclitaxel. Low amount of Mesenchymal Stromal Cells exerted only a little anticancer activity. CONCLUSION: Micro-fragmented fat inhibited mesothelioma cell proliferation in vitro and exerted a significant control of the mesothelioma xenograft growth in vivo.
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Mesotelioma Maligno , Mesotelioma , Humanos , Animales , Ratones , Xenoinjertos , Línea Celular Tumoral , Mesotelioma/tratamiento farmacológico , Mesotelioma/patología , Paclitaxel/farmacologíaRESUMEN
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.
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Antineoplásicos Fitogénicos/farmacología , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Células Madre Mesenquimatosas/efectos de los fármacos , Osteoblastos/citología , Paclitaxel/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Aspirina/farmacología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Condrocitos/citología , Condrocitos/metabolismo , Femenino , Humanos , Indometacina/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Osteoblastos/metabolismo , Ácido Valproico/farmacologíaRESUMEN
A new cationic Pt(II) complex bearing 8-aminoquinoline as chelating ligand (called Pt-8AQ) was evaluated against two human carcinomas, one mesothelioma, and three glioblastoma cell lines. The in vitro comparison to the clinically approved CisPt showed a minor activity of Pt-8AQ against carcinoma and mesothelioma, whereas a significant activity of Pt-8AQ was observed on the proliferation of the three glioblastoma cell lines (U87-MG IC50 = 3.68 ± 0.69 µM; U373-MG IC50 = 11.53 ± 0.16 µM; U138-MG IC50 = 8.05 ± 0.23 µM) that was higher than that observed with the clinically approved CisPt (U87-MG IC50 = 7.27 + 1.80 µM; U373-MG IC50 = 22.69 ± 0.05 µM; U138-MG IC50 = 32.1 ± 4.44 µM). Cell cycle analysis proved that Pt-8AQ significantly affected the cell cycle pattern by increasing the apoptotic cells represented by the sub G0/G1 region related with a downregulation of p53 and Bcl-2. Moreover, an NMR investigation of Pt-8AQ interaction with 9-EtG, GSH, and Mets7 excluded DNA as the main target, suggesting a novel mechanism of action. Our study demonstrated the high stability of Pt-8AQ after incubation at 37 °C and a significant antineoplastic activity on glioblastomas. These features also make Pt-8AQ a good candidate for developing a new selective advanced cell chemotherapy approach in combination with MSCs.
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Hepatocellular carcinoma (HCC) is poorly beneficiated by intravenous chemotherapy due to inadequate availability of drugs at the tumor site. We previously demonstrated that human micro-fragmented adipose tissue (MFAT) and its devitalized counterpart (DMFAT) could be effective natural scaffolds to deliver Paclitaxel (PTX) to tumors in both in vitro and in vivo tests, affecting cancer growth relapse. Here we tested the efficacy of DMFAT-PTX in a well-established HCC in nude mice. MFAT-PTX and DMFAT-PTX preparations were tested for anti-cancer activity in 2D and 3D assays using Hep-3B tumor cells. The efficacy of DMFAT-PTX was evaluated after a single-shot subcutaneous injection near a Hep-3B growing tumor by assessing tumor volumes, apoptosis rate, and drug pharmacokinetics in an in vivo model. Potent antiproliferative activity was seen in both in vitro 2D and 3D tests. Mice treated with DMFAT-PTX (10 mg/kg) produced potent Hep-3B growth inhibition with 33% complete tumor regressions. All treated animals experienced tumor ulceration at the site of DMFAT-PTX injection, which healed spontaneously. Lowering the drug concentration (5 mg/kg) prevented the formation of ulcers, maintaining statistically significant efficacy. Histology revealed a higher number of apoptotic cancer cells intratumorally, suggesting prolonged presence of PTX that was confirmed by the pharmacokinetic analysis. DMFAT may be a potent and valid new tool for local chemotherapy of HCC in an advanced stage of progression, also suggesting potential effectiveness in other human primary inoperable cancers.
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BACKGROUND: Malignant Pleural Mesothelioma (MPM) is an aggressive tumor that has a significant incidence related to asbestos exposure with no effective therapy and poor prognosis. The role of mesenchymal stromal cells (MSCs) in cancer is controversial due to their opposite effects on tumor growth and in particular, only a few data are reported on MSCs and MPM. METHODS: We investigated the in vitro efficacy of adipose tissue-derived MSCs, their lysates and secretome against different MPM cell lines. After large-scale production of MSCs in a bioreactor, their efficacy was also evaluated on a human MPM xenograft in mice. RESULTS: MSCs, their lysate and secretome inhibited MPM cell proliferation in vitro with S or G0/G1 arrest of the cell cycle, respectively. MSC lysate induced cell death by apoptosis. The efficacy of MSC was confirmed in vivo by a significant inhibition of tumor growth, similar to that produced by systemic administration of paclitaxel. Interestingly, no tumor progression was observed after the last MSC treatment, while tumors started to grow again after stopping chemotherapeutic treatment. CONCLUSIONS: These data demonstrated for the first time that MSCs, both through paracrine and cell-to-cell interaction mechanisms, induced a significant inhibition of human mesothelioma growth. Since the prognosis for MPM patients is poor and the options of care are limited to chemotherapy, MSCs could provide a potential new therapeutic approach for this malignancy.
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Ciclo Celular , Proliferación Celular , Supervivencia Celular , Mesotelioma Maligno/patología , Adolescente , Adulto , Anciano , Animales , Línea Celular Tumoral , Femenino , Humanos , Células Madre Mesenquimatosas , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Adulto JovenRESUMEN
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.
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Antígenos CD/metabolismo , Sangre Fetal/citología , Glicoproteínas/metabolismo , Proteínas de Homeodominio/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Péptidos/metabolismo , Células Madre Pluripotentes/metabolismo , Transactivadores/metabolismo , Antígeno AC133 , Biomarcadores/metabolismo , Diferenciación Celular , Células Cultivadas , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Inmunohistoquímica , Células Madre Pluripotentes/citología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Células Madre/farmacología , Ácido Valproico/farmacologíaRESUMEN
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.
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Ensayo de Unidades Formadoras de Colonias/métodos , Animales , Antiinflamatorios no Esteroideos/toxicidad , Antineoplásicos/uso terapéutico , Antineoplásicos/toxicidad , Trasplante de Médula Ósea/efectos adversos , Relación Dosis-Respuesta a Droga , Humanos , Indometacina/uso terapéutico , Indometacina/toxicidad , Dosificación Letal Mediana , Macrófagos/fisiología , Dosis Máxima Tolerada , Ratones , Ratas , Trasplante Heterólogo/efectos adversosRESUMEN
The COVID-19 epidemic represents an unprecedented global health emergency, further aggravated by the lack of effective therapies. For this reason, several clinical trials are testing different off-label drugs, already approved for other pathologies. Mesenchymal stem/stromal cells (MSCs) have been tested during the last two decades for the treatment of various pathologic conditions, including acute and chronic lung diseases, both in animal models and in patients. In particular, promising results have been obtained in the experimental therapy of acute respiratory distress syndrome, which represents the most threatening complication of COVID-19 infection. Furthermore, more recently, great interest has been devoted to the possible clinical applications of extracellular vesicles secreted by MSCs, nanoparticles that convey much of the biological effects and of the therapeutic efficacy of their cells of origin. This review summarizes the experimental evidence underlying the possible use of MSCs and of MSC-EVs in severe COVID-19 infection and underlines the need to evaluate the possible efficacy of these therapeutic approaches through controlled studies under the supervision of the Regulatory Authorities.
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Infecciones por Coronavirus/terapia , Vesículas Extracelulares , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Neumonía Viral/terapia , Neumonía/terapia , Animales , COVID-19 , Infecciones por Coronavirus/complicaciones , Humanos , Pandemias , Neumonía/etiología , Neumonía Viral/complicacionesRESUMEN
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.
RESUMEN
Mesothelioma is a rare lethal tumor of dogs and humans involving cavities of the body. Dogs are considered a model for new drugs and therapeutic methods since they present spontaneous diseases similar to humans. Microfragmented adipose tissue (MFAT) uploaded by paclitaxel (PTX) is a drug delivery medium providing slow release of chemotherapic drugs. A dog affected by pleural, pericardial, and peritoneal mesothelioma was treated by 17 intracavitary ultrasound-guided injections of MFAT-PTX over 22 months. A long-lasting improvement of general conditions was observed, treatment was well-tolerated, and no toxicity or hypersensitivity was reported. Pharmacokinetic (PK) data indicated low drug localization in the circulatory system and a tendency to enter or remain in the extravascular compartments of the body. Indeed, low levels of free-circulating drugs for a short time produced low toxicity, whereas, a higher intracavitary PTX concentration can have major pharmacological efficacy. To our knowledge, this is the first time that mesothelioma has been treated using such a procedure, and this should be considered as a novel therapeutic approach. The low systemic absorption suggests the possible role of MFAT-PTX for loco-regional/intratumoral therapy also useful in other types of tumors, and further investigation is warranted.