Mesenchymal stromal cells primed with paclitaxel provide a new approach for cancer therapy

Abstract

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.

Figure 1. PTX priming of hMSCs and SR4987 does not affect viability and induces anti-proliferative activity on MOLT-4.

Different concentrations of PTX (from 0.1 to 10.000 ng/ml) were added to culture of hMSCs (Fig. 1A) and mouse SR4987 (Fig. 1B). The cytotoxic activity was evaluated at 24 h of treatment; the effect on proliferation at 7 day of culture by a MTT assay. Optical Density measured in cultures of MSC that did not receive PTX were considered as 100% proliferation. Note that concentration of PTX up to 10.000 ng/ml did not affect either hMSCs or SR4987 cell viability. The small tables insert in the A and B indicate the IC₅₀ and IC₉₀ induced by PTX on hMSCs and SR4987 respectively. Both the conditioned media (CM) from PTX primed cells (hMSCsPTX-CM and SR4987PTX-CM) produced a dose dependent growth inhibition of MOLT-4 reported (Fig. 1C) as percent of that produced by CM from untreated cells (hMSCs-CM and SR4987-CM). Note that at 1∶16 dilution of both hMSCsPTX-CM and SR4987PTX-CM produced 100% growth inhibition equal to those obtained with 3.13 ng/ml of PTX. This biological assay was used to estimate PEC released by single PTX treated hMSC and its accumulation in the culture medium during the time (D). The histogram indicates PEC released by hMSCPTX at different times of culture. The curve indicates the PEC accumulation in the hMSCsPTX-CM. Note that each hMSCPTX releases around 1 pg of PEC in 24 h, reaching a maximal accumulation of around 1.7 pg after 144 h. Value are the mean ± standard deviation (SD) of five independent experiments.

Figure 2. PTX-F internalization and release by hMSCs.

The internalization of PTX-F was analyzed by confocal microscopy (A) in live hMSCs primed 1 (1) or 24 (24) h with PTX-F (green) and loaded with the Golgi specific marker BOPIPY®TR ceramide (red). Cells were also observed 24 h after washing step (24+24). PTX-F accumulates in cells and co-localizes with Golgi apparatus or Golgi-derived vesicles. Mask panel highlights the co-localization between PTX-F and BODIPY®TR ceramide showing white spots, that indicate those pixels in which both the fluorescent signals are detectable.. White lines represent the cell boundary and arrows indicate vesicles close to the cell membrane. Scale bar: 20 µm. The release of PTX in the hMSCsPTX-CM at 24 h was analysed by HPLC. The elution profile (B) was compared to that of pure PTX at 1.000 ng/ml (C). The figure reports a chromatogram profile of one typical experiment where hMSCsPTX-CM evidences a peak that clearly identified PTX and that was quantified on a PTX standard curve as 68.1 ng/ml. Figure 2D reports the profile of the hMSCs-CM cultured in the absence of PTX. The peak labeled as I.S. is the internal standard Cephalomannine added to all samples for the correct quantification of PTX.

Figure 3. Primed hMSCsPTX and SR4987PTX inhibit proliferation of different TC lines in vitro.

In (A) is shown the kinetics of growth inhibition induced by serial dilutions of hMSCsPTX-CM on T98G, DU145 and of SR4987PTX-CM on B16 which was compared with activity of different concentrations of PTX on the same TCs (B). The CM addition produced a strong anti-proliferative effect on all TCs tested in a dose dependent manner: 1∶16 and 1∶4 dilutions IC₅₀ and IC₉₀ growth inhibition on all TC lines respectively, corresponding to the PTX concentrations necessary to obtain IC₅₀ and IC₉₀ on the different tumor cells as reported in small table insert in (B). Inhibition of TCs proliferation was also obtained by a direct co-culture assay. Primed hMSCsPTX mixed, at different ratios (1∶100–1∶10–1∶1 MSCs/TCs), with MOLT-4 (C), T98G (D), DU145 (E) showed dose dependent capacity to block TCs proliferation evaluated in a MTT test at 7 days expressed as percent of OD measured for TCs cultured in control medium alone (CTR) or in presence of not primed hMSCs. SR4987PTX behaved like hMSCsPTX. However, even not primed SR4987 per se showed some anti-proliferative capacity on B16 melanoma at 1∶1 ratio (F). The histograms report the mean ± SD of three experiments with the statistical significance as follows: * ( p<0.05),** ( p<0.01) vs tumor cell growth (CTRL).

Figure 4. PTX and primed hMSCsPTX inhibit ECs proliferation in vitro.

PTX inhibits ECs proliferation (A). HUVECs and HMECs (2×10⁵) were cultured for 72 h in the presence of different concentration of PTX. The IC₅₀ was around 4.69 ng/ml of PTX and at 10 ng/ml PTX significantly blocked both HUVECs and HMECs proliferation, which at higher doses was cytotoxic. Similarly, the addition of hMSCsPTX-CM greatly inhibit HUVECs (B) and HMECs (C) proliferation. At 1∶2 dilution, hMSCsPTX-CM was cytotoxic, while at higher 1∶4 and 1∶8 dilutions significantly inhibit ECs proliferation. Inhibition of ECs proliferation was obtained by co-culture assay. HMSCsPTX co-cultured at 1∶1 and 1∶5 ratio (MSCs/ECs) was cytotoxic for both HUVECs (D) and HMECs (E). At 1∶10 ratio hMSCsPTX continued to inhibit ECs proliferation. Untreated control hMSCs did not affect ECs. In (F) photographs of culture of hMSCsPTX mixed 1∶5 with HMECs. Cells fixed and stained at different time intervals are shown. hMSCsPTX kill HMECs as early as 24 h after seeding. White arrows indicate the islands of HMECs still present in the culture. Note that after 72 h most of the HMECs seeded were killed and only hMSCsPTX remain in the culture (20× magnification). Bars in the figures are the means ± SD of three separate experiments done in triplicate. * p<0.05, **p<0.01 vs untreated hMSCs.

Figure 5. HMSCsPTX inhibit microvessel out-growth in vitro and reduce tumor growth in vivo.

In (A) and (B) rat aorta ring assay was used to test hMSCsPTX-CM microvessel growth inhibition. In (A) hMSCsPTX-CM at different dilutions added to rat aorta rings in the presence or in the absence of VEGFa induced a great reduction of capillary outgrowth compared to CTRL medium and to hMSCs-CM. VEGFa 20 ng/ml was used as positive controls (**p<0.01 vs hMSCs-CM). In (B) photographs show hMSCsPTX-CM (at 1∶2 dilution), which induced capillary regression as demonstrated by the presence of vessel rupture and necrotic zones (arrows) (magnifications 20×). In (C), (D) and (E) the effects of hMSCsPTX in vivo on tumor takes (C), on DU145 (D) and B16 (E) growth are shown. Around 0.4×10⁶ hMSCsPTX and control untreated hMSCs were mixed (at ratio 1∶5 hMSCs/TCs/) with 2×10⁶ DU145 or B16 and then injected subcutaneously (s.c.) into mice. Tumor volumes were calculated by measuring the tumor diameters taken every two days with a calibre. The co-injection of hMSCsPTX with DU145 or B16, produced a significant delay of tumor appearance (C) as well as a great reduction of DU145 (D) and B16 (E) tumor volume, whereas the co-injection with untreated hMSCs did not affect either DU145 or B16 volumes, nor did the injection of TCs mixed 5 minutes before injection with 2.000 ng/ml of PTX (see Table 1). The insert in (D) and (E) is the photo of tumors of control, hMSCsPTX and hMSCs treated mice at time of sacrifice. *p<0.05 and **p<0.01 vs TCs alone or vs hMSCs treated mice.