Original paperCancer therapyHuman CD14+ cells loaded with Paclitaxel inhibit in vitro cell proliferation of glioblastoma
Introduction
To develop more effective therapies against human glioblastoma (GB), recent years have seen clinical trials based on immunotherapy strategies involving the use of various cell types, including natural killer cells, cytotoxic T-lymphocytes, lymphokine-activated killer cells and dendritic cells (DCs) [1], [2]. Most recently immunotherapy using DCs has been a particular focus, although the singular role of CD14+ cells in GB is still debated. The myeloid-derived suppressor cell circulating in the peripheral blood (PB) of GB-affected patients has been reported to contribute to the cellular immunosuppression characteristic of GB [3]. It is known that population of myeloid-derived suppressor cells contains immature myeloid cells (progenitors of granulocytes, DCs and macrophages), and different phenotypes of these cells have been associated with different cancer types. For example, in patients with melanoma, it has been reported that immunosuppressive CD14+HLA-DR–/low cells accumulate in the peripheral blood [4], [5] and are able to directly affect T cells by mechanisms involving the release of different cytokines [6], [7], [8]. In an attempt to develop new strategies to circumvent the immunosuppression associated with GB, novel approaches have been designed using autologous DC-based vaccination considered a promising strategy to attack high-grade glioma [9], [10], [11], [12], [13]. Two clinical trials involving the use of DCs prepared in the Good Manufacturing Practice facility are under investigation at Neurological Institute C. Besta. These trials should provide information about the validity of the immunotherapeutic approach to improve efficacy when combined with conventional therapy for the treatment of patients with GB [14], [15].
Our preliminary clinical data seem to confirm literature showing that autologous DC-based vaccination is safe and can produce positive effects on patients with this dramatic disease [2], [16], [17]. Additionally, our results suggest that DCs, pulsed with GB tumor lysate, can potentiate the anti-tumor immune response as a result of a complex mechanism involving the modulation of cytokines in the tumor microenvironment, decrease of regulatory T cells and inhibition of tumor proliferation through tumor necrosis factor-alpha [12].
In addition to the use of DCs for GB immunotherapy, PB-derived CD14+ and DCs have been engineered for delivery anti-cancer molecules [18] because, such as mesenchymal stromal cells (MSCs), these cells have the capacity to home tumor microenvironment [19]. Thus, we recently demonstrated that human MSCs isolated from various tissue sources (such as bone marrow, fat and dermis) are able to uptake and release anti-cancer drugs without any genetic manipulation. More specifically, we found that PTX, a potent anti-proliferative drug that is widely used in cancer therapy [20], [21], can be delivered by MSCs and kill cancer cells when MSCs loaded with PTX are in close proximity [22], [23]. On the basis of these results, we here asked whether CD14+ cells and DCs once primed with PTX could behave like MSCs, that is, whether they could deliver and release the drug and, more important, kill GB cancer cells if located in their vicinity. Our results demonstrate for the first time that both CD14+ and DCs are able to incorporate and release PTX in a concentration high enough to kill GB cells in vitro. Our results suggest the possibility of using autologous DCs not only for immunotherapy but also to deliver anti-cancer drugs in GB patients.
Section snippets
Isolation and purification of CD14+ cells from PB
The study was approved by the local institutional review board of the Istituto Di Ricovero e Cura a Carattere Scientifico Foundation Neurological Institute C. Besta, Milan, Italy, and informed written consent was obtained from all volunteers. PB samples were obtained from six healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated from 100 mL of PB of each healthy subject by Ficoll-Hypaque density gradient centrifugation (GE Healthcare Bio-Sciences, Uppsala, Sweden).
Isolation and characterization of CD14+cells
Six healthy donors were enrolled in this study (mean age 39.7 ± 8.2 years). An average of 13.07 ± 3.81 × 107 PBMCs with a mean monocyte percentage of 13.31 ± 3.55% was obtained from 100 mL of PB of healthy subjects after Ficoll-Hypaque density gradient centrifugation. At least 13.07 × 107 of starting PBMCs were necessary to obtain 1.9 × 107 DCs at the end of the procedure. After immunomagnetic separation, the percentage of CD14+ cells was 89.33 ± 6.82%, and viable cells were 96.02 ± 3.01%.
CD14+
Discussion
The procedure for the uptake and release of PTX, previously setup in our laboratory by using MSCs from different sources (fat, bone marrow and dermis) demonstrated that the drug release was effective in both in vitro and in vivo tumor growth [22], [23], [29]. In this study the PTX priming procedure has been applied both to CD14+ and DCs, which were isolated, prepared and characterized according to the current methodology used in clinical trials [12], [15]. Preliminary observations verified that
Acknowledgment
This research was partially supported by the Associazione Italiana per la Ricerca sul Cancro (Project AIRC 12037). ricerca corrente Fondazione Besta (project 62, supervisor: Daniela Lisini).
Disclosure of interest: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.
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These authors contributed equally to this work.