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

Abstract

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.

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.