Human amniotic mesenchymal stromal cells (hAMSCs) as potential vehicles for drug delivery in cancer therapy: an in vitro study

Abstract

Introduction: In the context of drug delivery, mesenchymal stromal cells (MSCs) from bone marrow and adipose tissue have emerged as interesting candidates due to their homing abilities and capacity to carry toxic loads, while at the same time being highly resistant to the toxic effects. Amongst the many sources of MSCs which have been identified, the human term placenta has attracted particular interest due to its unique, tissue-related characteristics, including its high cell yield and virtually absent expression of human leukocyte antigens and co-stimulatory molecules. Under basal, non-stimulatory conditions, placental MSCs also possess basic characteristics common to MSCs from other sources. These include the ability to secrete factors which promote cell growth and tissue repair, as well as immunomodulatory properties. The aim of this study was to investigate MSCs isolated from the amniotic membrane of human term placenta (hAMSCs) as candidates for drug delivery in vitro.

Methods: We primed hAMSCs from seven different donors with paclitaxel (PTX) and investigated their ability to resist the cytotoxic effects of PTX, to upload the drug, and to release it over time. We then analyzed whether the uptake and release of PTX was sufficient to inhibit proliferation of CFPAC-1, a pancreatic tumor cell line sensitive to PTX.

Results: For the first time, our study shows that hAMSCs are highly resistant to PTX and are not only able to uptake the drug, but also release it over time. Moreover, we show that PTX is released from hAMSCs in a sufficient amount to inhibit tumor cell proliferation, whilst some of the PTX is also retained within the cells.

Conclusion: Taken together, for the first time our results show that placental stem cells can be used as vehicles for the delivery of cytotoxic agents.

Fig. 1

Characterization of human amniotic mesenchymal stromal cells (hAMSCs). Phenotype of unprimed (a) and paclitaxel (PTX)-primed (b) hAMSCs. The percentage of positive cells is indicated in each plot. Cell morphology is shown in panel c, magnification × 4. The images on the left show unprimed hAMSC (top) or hAMSC after the 24-hour treatment with 2,000 ng/ml of PTX (bottom). The images on the right show unprimed hAMSC (top) or hAMSC at the time at which conditioned media and lysates were collected and tested for their anti-proliferative activity against CFPAC-1 (bottom, 48 hrs)

Fig. 2

Paclitaxel (PTX) sensitivity of human amniotic mesenchymal stromal cells (hAMSCs). a Twenty-four-hour cytotoxicity assay of hAMSCs in the presence of PTX. Bars represent mean value ± SD for five donors. b Seven-day anti-proliferation assay of hAMSCs (seven donors) in the presence of 10-fold serial dilutions of PTX (linear regression analysis). c Paclitaxel half maximal inhibitory concentration (IC ₅₀) values (expressed as ng/ml) assessed by linear regression analysis in the anti-proliferation assay. For each donor, the mean value ± SD of at least two independent experiments is reported. The analysis of IC₅₀ values by a multiple comparison test showed that only the IC₅₀ of donor 5 is significantly different from mean value (*)

Fig. 3

Paclitaxel (PTX) uptake/release by human amniotic mesenchymal stromal cells (hAMSCs). a Release of PTX over time evaluated in four out of seven donors. Bars represent the amount of PTX (expressed as picograms/cell) released at each time point, and the curve expresses the total amount of PTX released over time. b Proliferation curves of CFPAC-1 in the presence of serial dilutions of PTX (white circles) or conditioned media from PTX-primed hAMSCs (hAMSCsPTX-CM) (black circles). Each point represents the mean value ± SD. The curve of hAMSCsPTX-CM represents mean values obtained from seven donors. The half maximal inhibitory concentration (IC ₅₀) and the volume of CM able to inhibit tumor growth by 50 % (V ₅₀) are shown. PEC paclitaxel equivalent concentration

Fig. 4

Evaluation of paclitaxel (PTX) internalized by human amniotic mesenchymal stromal cells (hAMSCs) but not released into culture medium. a Proliferation curves of CFPAC-1 in the presence of serial dilutions of conditioned media from PTX-primed hAMSCs (hAMSCsPTX-CM) (solid line) or lysates from PTX-primed hAMSCs (hAMSCsPTX-LYS) (dashed line). Five different hAMSC donors were tested. b The graph shows the amount of PTX incorporated and released by hAMSCs (CM) and the amount incorporated and retained inside the cells (LYS), expressed as percentages of the total incorporated PTX, considered 100 %. Bars represent the mean values ± SD. Five different hAMSC donors were tested. The difference between CM and LYS was not statistically significant (p >0.05)

Fig. 5

Effect of verapamil on paclitaxel (PTX) toxicity and PTX uptake/release by human amniotic mesenchymal stromal cells (hAMSCs). a P-glycoprotein (P-gp) expression is represented as the ratio of mean fluorescence intensity (MFI) for each donor: nd not determined. b Proliferation of hAMSCs in the presence of PTX and 20 μM verapamil (VP). Half maximal inhibitory concentration (IC₅₀) values (mean ± SD) were calculated by linear regression analysis. c Proliferation curves of CFPAC-1 in the presence of serial dilutions of PTX (white circles), conditioned media from PTX-primed hAMSCs (hAMSCsPTX-CM) (black circles) or hAMSCsPTX-CM collected from cells primed with PTX in the presence of 20 μM VP (black triangles). d Proliferation curves of CFPAC-1 in the presence of serial dilutions of hAMSCsPTX-CM (solid line) or hAMSCsPTX-LYS (dashed line) from PTX primed hAMSCs. Both CM and LYS were obtained from hAMSCs primed with PTX in the presence of 20 μM VP. e Amount of PTX incorporated and released by hAMSCs (CM) and the amount incorporated and retained inside the cells (LYS), expressed as percentages of the total incorporated PTX, considered 100 %. hAMSCs were primed in the presence of 20 μM VP. Bars represent the mean values ± SD. The difference between CM and LYS was not statistically significant (p >0.05). To evaluate the effect of VP, hAMSCs from two donors were used