Lab-on-Chip for Testing Myelotoxic Effect of Drugs and Chemicals

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.In the last twenty years, one of the main goals in the drug discovery field has been the development of reliable in vitro models. In particular, in 2006 the European Centre for the Validation of Alternative Methods (ECVAM) has approved the Colony forming Unit-Granulocytes-Macrophages (CFU-GM) test, which is the first and currently unique test applied to evaluate the myelotoxicity of xenobiotics in vitro. The present work aimed at miniaturizing this in vitro assay by developing and validating a Lab-on-Chip (LoC) platform consisting of a high number of bioreactor chambers with screening capabilities in a high-throughput regime

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

Uptake-release by MSCs of a cationic platinum(II) complex active in vitro on human malignant cancer cell lines

In this study, the in vitro stability of cisplatin (CisPt) and cationic platinum(II)-complex (caPt(II)-complex) and their in vitro activity (antiproliferative and anti-angiogenic properties) were investigated against three aggressive human tumor cell lines. caPt(II)-complex shown a high stability until 9 days of treatment and displayed a significant and higher activity than CisPt against both NCI-H28 mesothelioma (19.37 \ub1 9.57 \u3bcM versus 34.66 \ub1 7.65 \u3bcM for CisPt) and U87 MG glioblastoma (19.85 \ub1 0.97 \u3bcM versus 54.14 \ub1 3.19 for CisPt). Mesenchymal Stromal Cells (AT-MSCs) showed a significant different sensitivity (IC50=71.9 \ub1 15.1 \u3bcM for caPt(II)-complex and 8.7 \ub1 4.5 \u3bcM for CisPt) to the antiproliferative activity of caPt(II)-complex and CisPt. The ability of MSCs to uptake both the drugs in a similar amount of 2.49 pM /cell, suggested a possible development of new therapies based on cell mediated drug delivery

Automated Large-Scale Production of Paclitaxel Loaded Mesenchymal Stromal Cells for Cell Therapy Applications

Mesenchymal stromal cells (MSCs) prepared as advanced therapies medicinal products (ATMPs) have been widely used for the treatment of different diseases. The latest developments concern the possibility to use MSCs as carrier of molecules, including chemotherapeutic drugs. Taking advantage of their intrinsic homing feature, MSCs may improve drugs localization in the disease area. However, for cell therapy applications, a significant number of MSCs loaded with the drug is required. We here investigate the possibility to produce a large amount of Good Manufacturing Practice (GMP)-compliant MSCs loaded with the chemotherapeutic drug Paclitaxel (MSCs-PTX), using a closed bioreactor system. Cells were obtained starting from 13 adipose tissue lipoaspirates. All samples were characterized in terms of number/viability, morphology, growth kinetics, and immunophenotype. The ability of MSCs to internalize PTX as well as the antiproliferative activity of the MSCs-PTX in vitro was also assessed. The results demonstrate that our approach allows a large scale expansion of cells within a week; the MSCs-PTX, despite a different morphology from MSCs, displayed the typical features of MSCs in terms of viability, adhesion capacity, and phenotype. In addition, MSCs showed the ability to internalize PTX and finally to kill cancer cells, inhibiting the proliferation of tumor lines in vitro. In summary our results demonstrate for the first time that it is possible to obtain, in a short time, large amounts of MSCs loaded with PTX to be used in clinical trials for the treatment of patients with oncological diseases

Sensitivity of mesenchymal stromal cells to a new imidazole-based cationic Pt(II) complex with high in vitro anticancer activity

OBJECTIVE: Platinum drugs endowed with a novel chemical structure could offer an alternative therapeutic strategy, allowing to enlarge the spectrum of activity and to overcome the many drawbacks of the well-known cisplatin (CisPt) and its derivatives. Our group synthesised a new caPt(II)-complex that showed a very effective cytotoxic effect on triple-negative breast cancer cells and on cell lines partially resistant to cisplatin. In this study, we compared the in vitro stability of CisPt and caPt(II)-complex and their in vitro activity against human tumour cell lines. The drug sensitivity of Mesenchymal Stromal Cells (MSCs) and their ability to uptake and release the drugs was also investigated. MATERIALS AND METHODS: AT-MSCs were isolated, characterized and expanded from human adipose tissue. Drug stability was studied following incubation at 37\ub0C in complete cell culture medium both in the absence and in the presence of a monolayer of MSCs. The effect of CisPt and caPt(II)-complex was tested against mesothelioma (NCI-H28), glioblastoma (U87MG), pancreatic adenocarcinoma (CFPAC-1) and AT-MSCs by using a MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium) anti-proliferative assay in 96 multiwell plates. The amount of drugs incorporated and released by AT-MSCs drugs was evaluated by inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: We found that caPt(II)-complex had a high stability until 9 days of treatment while CisPt lost its anticancer activity after only 24 hours of treatment. CisPt was significantly more active (IC50= 9.64 \ub1 5.10 \ub5M) than caPt(II)-complex (IC50= 21.25 \ub1 6.68 \ub5M) on CFPAC1 proliferation. On the contrary, caPt(II)-complex showed a significant higher activity than CisPt both against NCI-H28 mesothelioma (19.37 \ub1 9.57 \ub5M versus 34.66 \ub1 7.65 \ub5M for CisPt) and U87 MG (19.85 \ub1 0.97 \ub5M versus 54.14 \ub1 3.19 for CisPt). AT-MSCs showed a sensitivity to the cytotoxic effect of caPt(II)-complex (IC50=92.8 \ub1 28.9 \ub5M) and CisPt (IC50= 93.5 \ub1 47.6 \ub5M) that does not differ significantly but with a higher variability of response to CisPt expressed by different donors of AT-MSCs. To the antiproliferative activity of caPt(II)-complex and CisPt, AT-MSCs showed a significant different sensitivity (IC50= 71.9 \ub1 15.1 \ub5M for caPt(II)-complex and 8.7 \ub1 4.5 \ub5M for CisPt). AT-MSCs are able to uptake both the drugs in a similar amount of 2.49 pM /cell. DISCUSSION AND CONCLUSION: The high stability of caPt(II)-complex together with its significant anticancer activity against mesothelioma and glioblastoma makes this new platinum derivative a very interesting molecule able to improve cancer chemotherapy. The low sensitivity of AT- MSCs to the antiproliferative action exerted by caPt(II)-complex together with their ability to uptake and release the drug will be further investigated in order to optimize the drug loading procedure and verify the possibility to set up a system of cell mediated delivery of caPt(II) complex

Pigmented nodular melanoma: the predictive value of dermoscopic features using multivariate analysis

BACKGROUND: Nodular melanoma (NM), representing 10-30% of all melanomas, plays a major role in global mortality related to melanoma. Nonetheless, the literature on dermoscopy of NM is scanty. OBJECTIVES: To assess odds ratios (ORs) to quantify dermoscopic features of pigmented NM vs. pigmented superficial spreading melanoma (SSM), and pigmented nodular nonmelanocytic and benign melanocytic lesions. METHODS: To assess the presence or absence of global patterns and dermoscopic criteria, digitized images of 457 pigmented skin lesions from patients with a histopathological diagnosis of NM (n = 75), SSM (n = 93), and nodular nonmelanocytic and benign melanocytic lesions (n = 289; namely, 39 basal cell carcinomas, 85 seborrhoeic keratoses, 81 blue naevi, and 84 compound/dermal naevi) were retrospectively collected and blindly evaluated by three observers. RESULTS: Multivariate analysis showed that ulceration (OR 4.07), homogeneous disorganized pattern (OR 10.76), and homogeneous blue pigmented structureless areas (OR 2.37) were significantly independent prognostic factors for NM vs. SSM. Multivariate analysis of dermoscopic features of NM vs. nonmelanocytic and benign melanocytic lesions showed that the positive correlating features leading to a significantly increased risk of NM were asymmetric pigmentation (OR 6.70), blue-black pigmented areas (OR 7.15), homogeneous disorganized pattern (OR 9.62), a combination of polymorphous vessels and milky-red globules/areas (OR 23.65), and polymorphous vessels combined with homogeneous red areas (OR 33.88). CONCLUSIONS: Dermoscopy may be helpful in improving the recognition of pigmented NM by revealing asymmetric pigmentation, blue-black pigmented areas, homogeneous disorganized pattern and abnormal vascular structures, including polymorphous vessels, milky-red globules/areas and homogeneous red areas

Mesenchymal Stromal Cells Primed with Paclitaxel Provide a New Approach for Cancer Therapy

BACKGROUND: Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation. METHODS AND PRINCIPAL FINDINGS: Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth. CONCLUSIONS: These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy

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 obtaine

Involvement of NOD2 in macrophage response to leishmania tropica infection

BACKGROUND-AIM Leishmaniasis is a protozoan disease caused by parasites belonging to the genus Leishmania. In the human host, these parasites invade macrophages where they develop into intracellular amastigotes and multiply within phagolysosomes. Host cells can control the infection initially through the triggering of innate immune responses. NOD-like receptors (NLRs) are a family of innate immune cytosolic receptors able to recognize pathogen-associated molecular patterns. NOD1 and NOD2 detect pathogens that are able to invade and multiply intracellularly. Once stimulated, these receptors induce the activation of NF-\u3baB and MAPKs, which lead to the transcription of genes involved in inflammation and immune responses. The aim of this work was to evaluate the role of NOD2 in some innate immune responses of macrophages infected with L. tropica. In particular, the production of TNF-a, or nitric oxide (NO), and the expression of inducible NO synthase (iNOS) were evaluated METHODS Immortalized bone marrow-derived macrophages (BMDM) from wild type (WT) C57Bl/6 or knockout (KO) mice for NOD2 were used. The levels of TNF-a released into the supernatants of BMDM-WT or -NOD2-KO treated with L. tropica were measured by ELISA. Also, the presence of nitrite was evaluated, through the Griess test, as an indication of nitric oxide (NO) production. Finally, protein and gene expression levels of iNOS were retrieved by Western blot analysis and realtime PCR, respectively. RESULTS The involvement of NOD2 in BMDM treated with L. tropica was elucidated as the levels of TNF-a or NO released from BMDM-WT infected with L. tropica were significantly higher compared to the BMDM-NOD2-KO. On the other side, the expression of iNOS (RNA and protein) was higher in BMDM-WT treated with L. tropica than in BMDM-NOD2-KO. CONCLUSIONS Altogether, these data indicated a crucial role of NOD2 for these innate immune responses of BMDM infected with L.tropica