Drug-releasing mesenchymal cells strongly suppress B16 lung metastasis in a syngeneic murine model

Abstract

Background: Mesenchymal stromal cells (MSCs) are considered an important therapeutic tool in cancer therapy. They possess intrinsic therapeutic potential and can also be in vitro manipulated and engineered to produce therapeutic molecules that can be delivered to the site of diseases, through their capacity to home pathological tissues. We have recently demonstrated that MSCs, upon in vitro priming with anti-cancer drug, become drug-releasing mesenchymal cells (Dr-MCs) able to strongly inhibit cancer cells growth.

Methods: Murine mesenchymal stromal cells were loaded with Paclitaxel (Dr-MCsPTX) according to a standardized procedure and their ability to inhibit the growth of a murine B16 melanoma was verified by in vitro assays. The anti-metastatic activity of Dr-MCsPTX was then studied in mice injected i.v. with B16 melanoma cells that produced lung metastatic nodules. Lung nodules were counted under a dissecting stereomicroscope and metastasis investigated by histological analysis.

Results: We found that three i.v. injections of Dr-MCsPTX on day 5, 10 and 15 after tumor injection almost completely abolished B16 lung metastasis. Dr-MCsPTX arrested into lung by interacting with endothelium and migrate toward cancer nodule through a complex mechanism involving primarily mouse lung stromal cells (mL-StCs) and SDF-1/CXCR4/CXCR7 axis.

Conclusions: Our results show for the first time that Dr-MCsPTX are very effective to inhibit lung metastasis formation. Actually, a cure for lung metastasis in humans is mostly unlikely and we do not know whether a therapy combining engineered MSCs and Dr-MCs may work synergistically. However, we think that our approach using Dr-MCs loaded with PTX may represent a new valid and additive therapeutic tool to fight lung metastases and, perhaps, primary lung cancers in human.

Fig. 1

Histological examination of the lungs showing the potent anti-metastatic activity of SR4987PTX treatment. a HE staining shows the presence of several B16 lung nodules (black arrows) in control mice (CTRL) and in SR4987 treated mice. Some metastasis are present in mice treated with a very high dose of PTX (10 mg/kg), while mice treated with SR4987PTX appear without metastatic nodules. b Fontana-Masson staining shows the presence of lung micrometastasis (red arrows) in PTX and their absence in SR4987PTX treated mice (boxes 600x magnifications)

Fig. 2

Adhesion of SR4987 and SR4987PTX to L-MECs and mL-StCs improved by B16-CM, TNFα and their association. a quantification of SR4987 and SR4987PTX that adhere to L-MECs monolayers not treated (CTRL) or treated with TNFα, B16-CM and TNFα + B16-CM respectively. b photographs (20x), showing the SR4987PTX cells (white arrows) that adhere on L-MEC monolayers. c quantification of SR4987 and SR4987PTX that adhere to mL-StCs monolayers. d photographs (20x) showing the adherent SR4987PTX cells on mL-StCs monolayer. The bars in the figure are the mean ± SD of cells counted with a calibrated eyepiece in 15 different fields at 40X magnification. Each test was run in quadruplicate. (*P < 0.05;**P < 0.01 vs CTRL)

Fig. 3

Chemotaxis of SR4987 and SR4987PTX induced by mL-StCs-CM upon priming with B16-CM or B16-CM + TNFα mediated by SDF-1/CXCR4/CXCR7 axis. In (a) SR4987PTX and (b) SR4987 cells migration. Bars are the mean ± SD of three independent experiments. Note the chemotactic activity of mL-StCs-B16-CM and mL-StCs-TNFα-B16-CM on both SR4987PTX and SR4987 (*P < 0.05;**P < 0.01 versus CTRL). B16-CM and mL-StCs-CM are not effective while SDF-1 induces SR4987PTX and SR4987 chemotaxis. In (c) SDF-1 secreted by the B16 melanoma cells and by mL-StCs under different stimuli. Bars are the mean value ± SD of SDF-1 detected in the CMs normalized for the same number of cells (10⁶). In (d) the analysis of SDF-1 at mRNA level in B16 and mLStCs under different culture conditions. Bars ± SD are the relative gene expression calculated by a comparative method (2^-ΔΔCt) using GAPDH as an housekeeping gene. In (e) SR4987PTX and (f) SR4987 cells migration in the presence of anti-SDF-1 (1 μg/ml) or after cells priming with AMD3100 (1 μM). Note that both anti-SDF-1 and AMD3100 significantly reduce chemiotaxis (° < 0.05 versus no anti-SDF-1 addition; **p < 0.01 versus no AMD3100 treatment)

Fig. 4

SDF-1 expression and presence of SDF-1+ cells in the lung of mice bearing B16-melanoma metastasis. a-f Immunostaining for SDF-1 of normal and metastatic lung. In (a) normal lung parenchyma shows no staining for SDF-1 at lower (200x left panel) and at higher magnification (600x right panel). In (b) metastatic lung parenchyma shows an intense SDF-1 staining at both magnifications (200x and 600x). In (c,d) the metastatic lung shows the presence of SDF-1+ cells (white arrows) in the parenchyma near or at periphery of metastasis (m). In (e,f) high magnification of a lung metastasis (m) showing the presence of SFD-1 + cells (white arrows) and a diffuse SDF-1 staining (red arrows) along the entire border of the metastasis

Fig. 5

Sca-1+ cells in lung parenchyma of SR4987PTX injected mice localize in vessels nearby metastatic nodule. Anti-Sca-1 antibodies were used to investigate the capacity of SR4987 and SR4987PTX to home lung B16 nodules. a quantification of Sca-1 + cells in the lungs. Bars are the mean ± SD of three mice and are the number of Sca-1⁺cells/mm² counted at 400X (**p < 0.01 vs CTRL or PTX treated mice). b,c,f,h Immunostaining for Sca-1 of metastatic lung parenchyma and (d,e,g,i) metastasis (m) area. Very few Sca-1+ cells (white arrows) are present in the lung of control and PTX treated mice in both parenchyma (b,c) and metastasis area (d,e). Mice treated either with SR4987 (f,g) and SR4987PTX (h,i) show many Sca-1+ cells in the lung parenchyma near the vessels (V) and at the periphery of metastatic nodules (200x magnifications). (J) Fontana-Masson staining showing B16 metastasis nearby (top left panel) or surrounding a lung microvessel (bottom right panel). In (L,M) Sca-1+ cells (black arrows) are attached to endothelium of tumor infiltrating microvessels