Giulio Alessandri

Glioblastoma-derived tumorospheres identify a population of tumor stem-like cells with angiogenic potential and enhanced multidrug resistance phenotype.

We investigated in vitro the properties of selected populations of cancer stem‐like cells defined as tumorospheres that were obtained from human glioblastoma. We also assessed their potential and capability of differentiating into mature cells of the central nervous system. In vivo, their tumorigenicity was confirmed after transplantation into the brain of non‐obese diabetic/severe combined immunodeficient (NOD‐SCID) mice. The angiogenic potential of tumorospheres and glioblastoma‐derived cells grown as adherent cells was revealed by evaluating the release of angiogenic factors such as vascular endothelial growth factor and CXCL12 by ELISA, as well as by rat aortic ring assay. The proliferative response of tumorospheres in the presence of CXCL12 was observed for the first time. Multidrug resistance‐associated proteins 1 and 3 as well as other molecules conferring multidrug resistance were higher when compared with primary adherent cells derived from the same tumor. Finally, we obtained cells from the tumor developing after grafting that clearly expressed the putative neural stem cell marker CD133 as shown by FACS analysis and also nestin and CXCR4. The cells positivity for glial fibrillary acidic protein was very low. Moreover these cells preserved their angiogenic potential. We conclude that human glioblastoma could contain tumor cell subsets with angiogenic and chemoresistance properties and that this chemoresistance potential is highly preserved by immature cells whereas the angiogenic potential is, to a higher extent, a property of mature cells. A better understanding of the features of these cell subsets may favor the development of more specifically targeted therapies.

Human CD133 + Progenitor Cells Promote the Healing of Diabetic Ischemic Ulcers by Paracrine Stimulation of Angiogenesis and Activation of Wnt Signaling

We evaluated the healing potential of human fetal aorta–derived CD133+ progenitor cells and their conditioned medium (CD133+ CCM) in a new model of ischemic diabetic ulcer. Streptozotocin-induced diabetic mice underwent bilateral limb ischemia and wounding. One wound was covered with collagen containing 2×104 CD133+ or CD133− cells or vehicle. The contralateral wound, covered with only collagen, served as control. Fetal CD133+ cells expressed high levels of wingless (Wnt) genes, which were downregulated following differentiation into CD133− cells along with upregulation of Wnt antagonists secreted frizzled-related protein (sFRP)-1, -3, and -4. CD133+ cells accelerated wound closure as compared with CD133− or vehicle and promoted angiogenesis through stimulation of endothelial cell proliferation, migration, and survival by paracrine effects. CD133+ cells secreted high levels of vascular endothelial growth factor (VEGF)-A and interleukin (IL)-8. Consistently, CD133+ CCM accelerated wound closure and reparative angiogenesis, with this action abrogated by coadministering the Wnt antagonist sFRP-1 or neutralizing antibodies against VEGF-A or IL-8. In vitro, these effects were recapitulated following exposure of high-glucose-primed human umbilical vein endothelial cells to CD133+ CCM, resulting in stimulation of migration, angiogenesis-like network formation and induction of Wnt expression. The promigratory and proangiogenic effect of CD133+ CCM was blunted by sFRP-1, as well as antibodies against VEGF-A or IL-8. CD133+ cells stimulate wound healing by paracrine mechanisms that activate Wnt signaling pathway in recipients. These preclinical findings open new perspectives for the cure of diabetic ulcers.

Paclitaxel is incorporated by mesenchymal stromal cells and released in exosomes that inhibit in vitro tumor growth: A new approach for drug delivery

Mesenchymal stromal cells (MSCs) have been proposed for delivering anticancer agents because of their ability to home in on tumor microenvironment. We found that MSCs can acquire strong anti-tumor activity after priming with Paclitaxel (PTX) through their capacity to uptake and then release the drug. Because MSCs secrete a high amount of membrane microvesicles (MVs), we here investigated the role of MVs in the releasing mechanism of PTX. The murine SR4987 line was used as MSC model. The release of PTX from SR4987 in the conditioned medium (CM) was checked by HPLC and the anti-tumor activity of both CM and MVs was tested on the human pancreatic cell line CFPAC-1. MVs were isolated by ultracentrifugation, analyzed by transmission (TEM) and scanning electron microscopy (SEM), and the presence of PTX by the Fourier transformed infrared (FTIR) microspectroscopy. SR4987 loaded with PTX (SR4987PTX) secreted a significant amount of PTX and their CM possessed strong anti-proliferative activity on CFPAC-1. At TEM and SEM, SR4987PTX showed an increased number of vacuole-like structures and shed a relevant number of MVs, but did not differ from untreated SR4987. However, SR4987PTX-derived-MVs (SR4987PTX-MVs) demonstrated a strong anti proliferative activity on CFPAC-1. FTIR analysis of SR4987PTX-MVs showed the presence of an absorption spectrum in the corresponding regions of the PTX marker, absent in MVs from SR4987. Our work is the first demonstration that MSCs are able to package and deliver active drugs through their MVs, suggesting the possibility of using MSCs as a factory to develop drugs with a higher cell-target specificity.

Human vasculogenesis ex vivo: embryonal aorta as a tool for isolation of endothelial cell progenitors.

Vasculogenesis, the de novo formation of new blood vessels from undifferentiated precursor cells or angioblasts, has been studied with experimental in vivo and ex vivo animal models, but its mechanism is poorly understood, particularly in humans. We used the aortic ring assay to investigate the angioforming capacity of aortic explants from 11- to 12-week-old human embryos. After being embedded in collagen gels, the aorta rings produced branching capillary-like structures formed by mesenchymal spindle cells that lined a capillary-like lumen and expressed markers of endothelial differentiation (CD31, CD34, von Willebrand factor [vWF], and fms-like tyrosine kinase-1 [Flk-1;[sol;vascular endothelial growth factor receptor 2 [VEGFR2]). The cell linings of these structures showed ultrastructural evidence of endothelial differentiation. The neovascular proliferation occurred primarily in the outer aspects of aortic rings, thus suggesting that the new vessels mainly arose from immature endothelial precursor cells localized in the outer layer of the aortic stroma, ie, a process of vasculogenesis rather than angiogenesis. The undifferentiated mesenchymal cells (CD34+/CD31−), isolated and cultured on collagen-fibronectin, differentiated into endothelial cells expressing CD31 and vWF. Furthermore, the CD34+/CD31+ cells were capable of forming a network of capillary-like structures when cultured on Matrigel. This is the first reported study showing the ex vivo formation of human microvessels by vasculogenesis. Our findings indicate that the human embryonic aorta is a rich source of CD34+/CD31− endothelial progenitor cells (angioblasts), and this information may prove valuable in studies of vascular regeneration and tissue bioengineering.

Mesenchymal Stem/Stromal Cells: A New "Cells as Drugs" Paradigm. Efficacy and Critical Aspects in Cell Therapy

Mesenchymal stem cells (MSCs) were first isolated more than 50 years ago from the bone marrow. Currently MSCs may also be isolated from several alternative sources and they have been used in more than a hundred clinical trials worldwide to treat a wide variety of diseases. The MSCs mechanism of action is undefined and currently under investigation. For in vivo purposes MSCs must be produced in compliance with good manufacturing practices and this has stimulated research on MSCs characterization and safety. The objective of this review is to describe recent developments regarding MSCs properties, physiological effects, delivery, clinical applications and possible side effects.

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.

Genetically Engineered Stem Cell Therapy for Tissue Regeneration

Abstract: Therapeutic angiogenesis/vasculogenesis represents a new approach to treat patients with ischemic disease not curable with conventional treatment. This review focuses on the rationale and preliminary results of combining stem cell and gene therapy for regenerative medicine. Under disease conditions, impaired neovascularization results from diminished vascular growth factor production and primary dysfunction of endothelial cells and their progenitors. Advances in our ability to genetically manipulate cells ex vivo has provided the technological platform to implement stem cell biology and circumvent the potential hazard of direct gene transfer. Ex vivo engineered endothelial progenitor cells have been used for the treatment of peripheral limb ischemia. The approach eliminates the drawback of immune response against viral vectors and makes feasible repeating the therapeutic procedure in case of injury recurrence. The strategy of using stem cells as vectors for curative agents proved to be of value for the treatment of pulmonary hypertension and thrombosis. Transplantation of neural stem cells genetically modified to secrete nerve growth factor was able to ameliorate the death of striatal projection neurons caused by transient focal ischemia in the adult rat. By a similar approach, engineered neural stem cells might be used for treating neurodegenerative disorders. Therefore, genetic manipulation of stem cells opens new avenues for regenerative medicine.

Characterization of tumor lines derived from spontaneous metastases of a transplanted murine sarcoma

Abstract Cell lines were obtained from nine individual spontaneous metastases of the murine mFS6 sarcoma by s.c. inoculation of lung deposits in syngeneic mice. When inoculated i.m., two lines (M4 and M7) from metastases had greater, and two (M8 and M9) had less metastasizing capacity than the primary mFS6 sarcoma. The remaining cell lines gave spontaneous metastases similar in incidence, number and weight to the primary mFS6 tumor. When inoculated i.v., M2 and M4 gave consistently more lung deposits than mFS6 cells, whereas cells from M1, M5, M6 and M8 produced fewer artificial metastases than the primary tumor. Eight individual lung metastases were disaggregated and tested for metastasizing capacity immediately after isolation from the lung parenchyma: spontaneous and artificial metastases from metastatic cells were similar to the primary tumor, except for one secondary which was less metastatic. The immunological characteristics of cell lines from metastases were studied by in vivo immunization and challenge. Seven lines from metastases and the primary tumor had weak cross-reacting transplantation antigens whereas two lines (M3 and M4) had no demonstrable antigenicity. Thus, cell lines from individual spontaneous metastases are heterogeneous in many respects, including metastasizing capacity. However in this model metastasis does not appear to be the expression of strong selection of variant cells with increased metastatic potential.

Human neuroblastoma cells produce extracellular matrix-degrading enzymes, induce endothelial cell proliferation and are angiogenic in vivo.

Direct experimental evidence shows that tumor growth and metastases are angiogenesis‐dependent. Neuroblastoma (NB) is the most common extracranial malignant solid tumor of childhood. In this study, we investigated 2 human NB cell lines, LAN‐5 and GI‐LI‐N, for their capacity to secrete 2 extracellular matrix‐degrading enzymes, MMP‐2 and MMP‐9, and to induce <0R><0B>in vitro<0R><0B> human microvascular endothelial cells (EC) to proliferate and in vivo angiogenesis in the chick embryo chorio‐allantoic membrane (CAM) assay. Conditioned medium (CM) from both cell lines stimulated in vitro EC proliferation and the effect of LAN‐5 CM was higher than that of GI‐LI‐N cells. Moreover, anti‐VEGF, but not anti‐FGF2 antibodies, prevented growth increment of EC. NB cell lines secreted the active form of MMP‐2 almost exclusively, LAN‐5 cells more than GI‐LI‐N cells. Both cell lines, LAN‐5 cells more than GI‐LI‐N ones, induced angiogenesis in the CAM assay. Our data suggest that the 2 NB cell lines are angiogenic, to LAN‐5 cells more than GI‐LI‐N ones. LAN‐5 cells are indeed endowed with a more aggressive and invasive phenotype. Int. J. Cancer 77:449–454, 1998.

Inhibition of neuroblastoma‐induced angiogenesis by fenretinide

Retinoids are a class of natural or synthetic compounds that participate in the control of cell proliferation, differentiation and fetal development. The synthetic retinoid fenretinide (HPR) inhibits carcinogenesis in various animal models. Retinoids have also been suggested to be effective inhibitors of angiogenesis. The effects of HPR on certain endothelial cell functions were investigated in vitro, and its effects on angiogenesis was studied in vivo, by using the chorioallantoic membrane (CAM) assay. HPR inhibited vascular endothelial growth factor‐ (VEGF‐) and fibroblast growth factor‐2‐ (FGF‐2)‐induced endothelial cell proliferation without affecting endothelial motility; moreover, HPR inhibited growth factor‐induced angiogenesis in the CAM assay. Furthermore, a significant antiangiogenic potential of HPR has also been observed in neuroblastoma (NB) biopsy‐induced angiogenesis in vivo. We previously demonstrated that supernatants derived from NB cell lines stimulated endothelial cell proliferation. In the present study, we found that this effect was abolished when NB cells were incubated in the presence of HPR. VEGF‐ and FGF‐2‐specific ELISA assays, performed on both NB cells derived from conditioned medium and cellular extracts, indicated no consistent effect of HPR on the level of these angiogenic cytokines. Moreover, RT‐PCR analysis of VEGF and FGF‐2 gene expression confirmed the above lack of effect. HPR was also able to significantly repress the spontaneous growth of endothelial cells, requiring at least 48–72 hr of treatment with HPR, followed by a progressive accumulation of cells in G1 at subsequent time points. Finally, immunohistochemistry experiments performed in the CAM assay demonstrated that endothelial staining of both VEGF receptor 2 and FGF‐2 receptor‐2 was reduced after implantation of HPR‐loaded sponges, as compared to control CAMs. These data suggest that HPR exerts its antiangiogenic activity through both a direct effect on endothelial cell proliferative activity and an inhibitory effect on the responsivity of the endothelial cells to the proliferative stimuli mediated by angiogenic growth factors.