Drug-releasing mesenchymal cells strongly suppress B16 lung metastasis in a syngeneic murine model
- Augusto Pessina†1Email author,
- Carlo Leonetti†2,
- Simona Artuso2,
- Anna Benetti3,
- Enrico Dessy4,
- Luisa Pascucci5,
- Daniela Passeri6,
- Augusto Orlandi6,
- Angiola Berenzi3,
- Arianna Bonomi1,
- Valentina Coccè1,
- Valentina Ceserani7,
- Anna Ferri7,
- Marta Dossena7,
- Pietro Mazzuca8,
- Emilio Ciusani9,
- Piero Ceccarelli5,
- Arnaldo Caruso8,
- Nazario Portolani10,
- Francesca Sisto1,
- Eugenio Parati7 and
- Giulio Alessandri7
© Pessina et al. 2015
Received: 22 June 2015
Accepted: 4 August 2015
Published: 13 August 2015
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.
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.
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.
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.
Adult mesenchymal stromal cells (MSCs) are a suitable cell source for cell-based therapies . MSCs possess intrinsic therapeutic potential , however, they can be in vitro manipulated and engineered to produce therapeutic molecules [3–6] that can be delivered to the site of disease, through their capacity to home pathological tissues [7–9].
Despite the molecular mechanisms involved in MSCs homing, the number of cells that reach a disease area may depend on the route of their administration [10, 11]. After intravenous (i.v) injection most of the cells remain trapped in draining organs such as lung, kidney and liver  and this may drastically limit the number of MSCs that can reach pathological area in other organs. However, lung, besides its draining capacity, has been shown a promise tissue target for cell-based therapies [13, 14].
Previous data from our group have shown that MSCs can acquire strong anti-cancer activity upon in vitro exposure to very high dose of chemotherapeutic agent such as Paclitaxel (PTX). In particular, we have shown that in vivo the co-injection as well as the intra-tumor injection of MSCs loaded with PTX strong inhibit tumor growth [15, 16]. MSCs become drug-releasing mesenchymal cells (Dr-MCs) because they are able to uptake and release the drug and thus kill tumor cells when located nearby. This property seems present not only in MSCs derived from bone marrow (BM) but also in those derived from adipose tissue  or in fibroblasts  and even in monocytes .
In order to expand our previous work, we here investigated the capacity of Dr-MCs loaded with PTX to inhibit lung metastasis induced by i.v. injection of B16 melanoma cells in syngeneic C57Bl6 mice. The Dr-MCs here used were the syngeneic SR4987 a MSC line developed from BM of BDF/1 mice . We found that SR4987 loaded with PTX (SR4987PTX) i.v. injected after tumor cells administration, abolished almost all lung metastasis. We demonstrated that SR4987PTX once in lung, accumulate nearby metastatic foci through a complex mechanism involving resident mouse lung stromal cells (mL-StCs), Stromal-Derived Factor 1 (SDF-1) and its receptors CXCR4/CXCR7. We propose to use Dr-MCs loaded with PTX as new therapeutic approach for lung metastasis.
Materials and methods
The murine melanoma cell line B16  and MOLT-4 (human acute T-lymphoblastic leukemia)  were provided by Centro Substrati Cellulari, ISZLER (Brescia, Italy). B16 cells were maintained in RPMI 1640 medium (Euroclone, UK) supplemented with 10 % fetal bovine serum (FBS) (LONZA Walkersville MD USA). MOLT-4 cells were cultured in complete IMDM +10 % FBS. The Mesenchymal stromal cell line SR4987 was established from a long-term BM-derived cell culture of BDF/1 mice (ATCC CRL-2028) . SR4987 cells were cultured in IMDM supplemented with 5 % FBS. SR4987 are positive for stem cell marker Vimentin, CD44+, CD73+, CD105+, CD106+, Sca-1+, CD34+, and CD45+ and have the capacity to differentiate into osteoblasts and chondrocytes . SR4987 cells were also transduced with green fluorescent protein (GFP) using lentiviral vector ( pCCLsin.PPThPGK.GFPpre) as previously described .
Human lung-derived microvascular endothelial cells (L-MECs) were obtained from Dr. Arnaldo Caruso (Laboratory of Microbiology, University of Brescia, Brescia, Italy). L-MECs were routinely maintained in endothelial basal growth medium (EBM-GM) (EGM bullet kit, LONZA Walkersville MD USA) on Collagen + Fibronectin coated T25 flasks.
Mouse lung stromal cells (mL-StCs) were freshly isolated from C57Bl6 mice by using 0.25 % collagenase D (Boehringer Mannheim Germany). The adherent mL-StCs were cultured in IMDM +5 % FBS and analyzed for mesenchymal markers CD44, CD29 and CD90 (all purchased from Abcam Cambridge UK, mouse MSCs panel) to confirm their mesenchymal origin.
PTX priming of SR4987 cells
PTX was purchased from Adipogen (Vinci-Biochem, Italy). The activity of PTX on SR4987 cells was determined in a 24 h (cytotoxicity test) and in a 7 day MTT assay (anti-proliferative test) as previously described . Priming of SR4987 with PTX (SR4987PTX) was carried out using PTX at 2,000 ng/ml as previously described .
In vitro antitumor assay
The effect of conditioned medium (CM) from SR4987 and SR4987PTX cells on B16 proliferation was evaluated by MTT assay . The inhibitory concentrations (IC50 and IC90) were determined according to the Reed and Muench formula . The antitumor activity of CM from SR4987PTX cells was compared to that of pure PTX and expressed as PTX equivalent concentration (PEC) . The PTX released by a single cells was calculated dividing PEC for the number of cells seeded and expressed as pg/cell. To establish the optimal dose of SR4987PTX to inject, co-cultures with B16 cells were made by using transwell inserts (0.4 μm pore size) (Becton Dickinson, USA) [15, 16].
Rosette adherence assay and Transmission electron microscopy (TEM) analysis
To study the interaction between B16 cells and SR4987, a rosette test was performed . B16 were mixed in a conical tube with SR4987 or SR4987PTX cells, in 500 μl of IMDM + 5 % FBS (ratio B16/SR4987 5:1). After 24 h of incubation at 37 °C in air + 5 % CO2, 20 μl of cells were collected by a micropipette and then transferred on a slide to evaluate rosette formation under an inverted microscope. Rosette were then analyzed by TEM as previously described .
Adhesion of SR4987 and SR4987PTX to L-MECs and to mL-StCs were performed following a previously described procedure . L-MECs and mL-StCs monolayers were stimulated by adding B16 derived CM (B16-CM), tumor necrosis factor-alpha (TNFα; 10 ng/ml; Sigma Chemical) and their combination for 24 h. L-MECs and mL-StCs were allowed to interact for 30’ with SR4987 and SR4987PTX (104 cells/well) in IMDM +0.2 % BSA. Each test was run in quadruplicate (see Additional file 1).
Transwell supports were used to test spontaneous migration and chemotaxis of SR4987 and SR4987PTX. Migration assay was performed as previously described . Test samples consisting in: mouse SDF-1 (1-100 ng/ml) (R&D system), TNFα (1-50 ng/ml), B16-CM (1:1-1:100 dilutions) mL-StCs-CM (1:1-1:100 dilutions), CM derived from mL-StCs primed for 24 h either with B16-CM (1:1) (mL-StCs-B16-CM), TNFα 10 ng/ml (mL-StCs-TNFα-CM) or B16-CM + TNFα (mL-StCs-B16-TNFα-CM), were placed in the lower compartment. The blocking effect of monoclonal antibodies (mAbs) anti mouse SDF-1 (R & D System), as well control mouse IgG, (0.01-1ug/ml) was tested by adding mAbs to medium containg SDF-1 or to CMs. To block SDF-1 receptors CXCR4/CXCR7, SR4987 and SR4987PTX (5×105) were pre-incubated for 1 h at 37 °C in IMDM + 0.2 % BSA in presence of different concentration of AMD3100 (0.1-10 μM) (purchased from Sigma). Each determination was done in duplicate (see Additional file 1).
Immunoassay for mouse SDF-1
CM samples recovered from 48 h cultured SR4987, SR4987PTX, B16 cells, or from mL-StCs stimulated or not with B16-CM, TNFα and B16-CM + TNFα were analyzed for the presence of SDF-1 using an Elisa kit (R&D System). The value of SDF-1 detected in the CM were normalized for the same number of cells counted at the end of incubation.
Flow Cytometry and PCR
The expression of Sca-1, CXCR4 and CXCR7 on SR4987 was determind by flow cytometry (FC) using the following antibodies: anti-Sca-1 FITC, anti-CXCR4 (ab1670) and anti-CXCR7 (all purchased from Abcam Cambridge UK). 20,000 events were acquired for each analysis using Epics “XL-MLC” (Beckman Coulter, USA) and histogram elaboration was performed with EXPO 32 software.
For the analysis of mRNA levels 1000 ng of total RNA isolated using the RNeasy kit (Qiagen) was reverse-transcribed using iScript cDNA Synthesis Kit (Bio-Rad Laboratories). Triplicate polymerase chain reactions were carried out on an CFX 96 Touch Real Time PCR Detection System (Bio-Rad Laboratories). Relative gene expression was calculated by a comparative method (2-ΔΔCt) using GAPDH as an housekeeping gene. Primers sequences were designed using Primer3 software.
In vivo experiments and Immunohistochemistry
Six-to eight-week old male C57BL/6 mice were purchased from Charles River (Calco, Milan, Italy). All the animal experiments were performed at the Animal Facility of Regina Elena National Cancer Institute in Rome Italy following directive 2010/63/EU. Healthy mice (2 each group) receiving i.v. injection of saline, 106 SR4987GFP and 106 SR4987GFP-PTX. Fluorescent cells were used to confirm their capacity to arrest into the lung. Mice were sacrificed at 6 h, 24 h and 48 h after treatments and lungs were minced and digested by collagenase (for 2 h at 37 °C) to obtain a cell suspension that was rapidly analyzed under Fluorescent microscopy. To study the anti-metastatic activity of SR4987PTX, mice were injected i.v. with 2.5 × 105 B16 melanoma cells /0.2 ml. On day 5, after B16 injection, mice (12 each group) were treated i.v. with 0.2 ml saline (controls), PTX 10 mg/Kg, SR4987 (105/0.2 ml) and SR4987PTX at 105 and 5×104/0.2 ml. Treatments were repeated on day 10 and 15. On day 21 after B16 injection, mice were euthanized, lungs removed and fixed in Bouin’s solution. Lung nodules were counted under a dissecting stereomicroscope. Metastasis were also investigated by histology using Hematoxylin and Eosin (HE) and by Fontana-Masson staining to detect micrometastasis . To investigate the capacity of SR4987 and SR4987PTX to home lung B16 nodules, mice (3 each group) were injected i.v. with 106 B16 cells. Upon 14 days mice were i.v. treated with saline (controls), PTX (10 mg/kg), SR4987 (105) and SR4987PTX (105) and sacrificed 48 h after treatments. The lungs were processed by immunohistochemistry for Sca-1 + cells detection by using rabbit monoclonal anti-Sca1/Ly6A/E antibody (1:150, Abcam) and polyclonal anti-SDF-1 (Santa Cruz Biotechnology Inc USA) for SDF-1 expression according to standard protocols [26, 27]. The number of Sca-1+cells was evaluated at high power field (HPF), 400X magnification. At least 10 fields for each case were randomly evaluated with variability less than 5 %.
Data are expressed as the mean ± standard deviation (SD). Differences between mean values were evaluated according to Student’s t-test performed by GRAPHPADINSTAT program (GraphPad Software Inc., San Diego, CA, USA). p values < 0.05 were considered statistically significant.
SR4987 localized in the lung after i.v. injection
In preliminary experiments we verified if mouse SR4987 and SR4987PTX cells localized in the lung after i.v injection. At this purpose, GFP transfected SR4987 cells (SR4987GFP) were prepared . At 6 h, 24 h, and 48 h following cells injection, mice were sacrificed and lungs analyzed for the presence of SR4987GFP. Both groups of mice treated with cells retained in the lung significant amount of GFP+ cells until 48 h upon injection. Maximal presence of cells was observed between 6 and 24 h. The viability of the GFP+ cells arrested in lungs was confirmed by culturing the cells obtained after lungs digestion. The presence of injected cells in the lung were also ascertained by evaluating Sca-1, a marker that is considered specific for murine MSCs  and highly expressed on SR4987 (Additional file 2: Figure S2A). A significant presence of Sca-1+ cells in both SR4987 and SR4987PTX treated mice were found, while rare Sca-1+ cells were detected in untreated mice (Additional file 2: Figure S2B) by indicating that physiological Sca-1 positive cells with bone marrow origin is rarely seen.
SR4987PTX bind B16 melanoma cells and inhibit their in vitro proliferation
In previous studies we have shown that SR4987 cells behaved as Dr-MCs since are very resistant to PTX cytotoxicity and able to uptake/release the drug [15, 16]. The quantification of PTX released by SR4987PTX was performed by testing the anti-proliferative activity of CM (SR4987PTX-CM) on MOLT-4, a leukemia cell line very sensitive to PTX . SR4987PTX release around 0.34 ± 0.01 pg and incorporate around 2.5 ± 0.05 pg of PTX/cell in 24 h . We found that SR4987PTX-CM inhibited B16 proliferation in a dose-dependent manner, with an IC50 at dilution 1/9.98 ± 2.4 corresponding at 7.18 ± 4.99 ng/ml PTX (all results are summarized in Additional file 2: Figure S3). To establish the dose of SR4987PTX cells to inject in vivo, we co-cultured SR4987PTX and B16 cells at different ratio using trans-well inserts. We found that the optimal dose/ratio of SR4987PTX/B16 was around 1/5 that inhibited B16 melanoma proliferation of 70 % (Additional file 2: Figure S4).
SR4987 and SR4987PTX were also able to bind B16 cells by a rosette formation assay. The ultrastructure analysis of the rosette showed the presence of junctional-like structure and even a gap-like junction between SR4987 and B16 cells. At TEM, the potent cytotoxic activity of SR4987PTX on B16 cells was very evident; most of the B16 cells bound to SR4987PTX were apoptotic and necrotic (Additional file 2: Figure S5).
In vivo systemic administration of SR4987 PTX suppresses B16 lung metastasis in syngeneic mice
SR4987PTX injected i.v. suppress B16 lung metastasis
days of treatments
Median number of lung metastasis (interval)
% of Metastasis inhibition
PTX (10 mg/Kg)
PTX (10 mg/Kg)
B16-CM and TNFα improve adhesion of SR4987PTX to microvascular endothelium and to stromal cells of the lung
Priming of mL-StCs with B16-CM alone or combined with TNFα stimulate SR4987PTX chemotaxis that is mediated by SDF-1/CXCR4/CXCR7 axis
Since the efficacy of SDF-1 and because SR4987 cells expressed a high level of CXCR4 (90 %) and, to minor extent, CXCR7 (20 %) SDF-1 receptors  (Additional file 2: Figure S7), we then verified if SDF-1 could be a mediator of SR4987 and SR4987PTX chemotaxis in the metastatic lung. We first analyzed in vitro the capacity of B16 and mL-StCs to release SDF-1. We found that B16 cells and mL-StCs did not release a significant amount of SDF-1 in the CM (Fig. 3c). However, when mL-StCs were primed with B16-CM alone or combined with TNFα the concentration of SDF-1 in the medium was significantly increased (Fig. 3c). These results were also confirmed by analyzing the expression of SDF-1 by PCR performed on mL-StCs under the different treatments conditions (Fig. 3d).
Migration experiments of SR4987PTX and SR4987 were repeated by adding anti-SDF-1 antibodies or in the presence of AMD3100 a molecule able to bind specifically the SDF-1 receptors CXCR4 and CXCR7 . Anti-SDF-1 antibodies and AMD3100 were both able to significantly reduce chemotaxis (Fig. 3e and f).
Increment of SDF-1 expression and presence of SDF-1+ cells in the lung of mice bearing B16 melanoma metastasis
SR4987PTX cells localize preferentially in the vessels nearby metastatic nodule
MSCs may represent a novel therapeutic tool for fighting cancer . MSCs can be engineered to produce and delivery anti-cancer molecules [3–6, 35, 36] or, as our group have recently demonstrated, MSCs can become Dr-MCs upon priming with anti-cancer drug like PTX, acquiring strong anti-cancer activity both and in vivo [15, 16]. Paclitaxel is an important molecule active on many solid tumors including melanoma  and also with anti-angiogenic activity. PTX priming of MSCs induces mitotic arrest of the cells without affecting their viability and allowing to the uptake/release mechanism to properly works. These features have been verified also for Vincristine and drugs with other mechanism of action as Gemcitabine and Doxorubicin. In any case, regardless the kind of manipulation used, it is necessary that MSCs maintain their homing ability because is crucial to localize their cytotoxic cargo in the tumor. At this regard, the therapeutic localization of MSCs in organs such as lung is certainly facilitated by i.v. administration [10, 11].
To expand our previous work, we here investigated whether the i.v. injection of Dr-MCs loaded with PTX may inhibit the formation of lung metastases, induced in C57Bl6 mice upon i.v. injection of syngeneic B16 melanoma cells. To exclude any immunological interference by the host, as Dr-MCs, we used the SR4987 cells, a BM-derived MSCs line syngeneic to C57Bl6 mice  and able to uptake and release PTX and to kill cancer cells [15, 16].
We found that 5×104 SR4987PTX cells injected i.v. three times, starting from day 5 after tumor injection, were sufficient to produce up to 90 % of lung metastasis inhibition; an effect that was even superior of those obtained with PTX given at the MTD of 10 mg/Kg. Because SR4987 may incorporate at maximum around 2.5 pg/cell of PTX , the dose of PTX that mice received through the cells was about 2000 times less than the groups treated with PTX. Thus, we conclude that the delivery of the drug through the cells has a significantly higher therapeutic index than the PTX given through the standard therapy. Previous reports have shown the efficacy of engineered MSCs delivering anti-cancer molecules, or MSCs per se, to inhibit lung metastases upon i.v. administration [5, 6, 38–40]. Our results expand these observations, and demonstrate for the first time that syngeneic MSCs loaded with PTX given systemically in mice bearing lung metastasis, have a potent therapeutic effect.
The capacity of SR4987PTX to home malignant lung nodules was investigated both in vitro and in vivo. In summary our results indicate that SR4987PTX once injected i.v., are able to reach metastatic lung nodules through a complex mechanism involving mainly the chemokine SDF-1/CXCL12 and its receptors CXCR4/CXCR7. These data confirm previous reports showing the importance of SDF-1/CXCR4/CXCR7 axis in the homing of MSCs to cancer environment [41–44]. However, besides this, our study highlights a new undiscovered mechanism of MSCs homing in metastatic lung that is not mediated directly by B16 cells but through the implication of lung StCs. Indeed, our in vitro data show that B16-CM neither stimulates chemotaxis of both SR4987 and SR4987PTX nor contains significant amount of SDF-1. In contrast, CM derived from mL-StCs primed with B16-CM, secretes SDF-1 and stimulates chemotaxis of SR4987PTX. Further, the in vivo analysis of the SDF-1+ cells localization in metastatic lung, reveals that most of the SDF-1+ cells are not present inside metastasis but are localized in the lung stroma or at the extreme periphery of metastatic nodules. We therefore conclude that SR4987PTX, once arrested in the lung, migrate toward B16 metastasis by an “indirect mechanism” mediated by SDF-1 secreted by StCs of the lung under the influence of factors released by B16 cancer cells. In this contest, TNFα seems to play an important role as enhancer of the B16 cancer cells activity.
Our results thus highlight the importance of StCs surrounding the metastatic nodule that seem to behave like tumor stromal cells in favoring the progression of primary cancer [45–47]. Whether this StCs-mediated attraction of MSCs is occurring in other metastatic organs besides lung, remains to be established. The finding that lung contains specific resident MSCs that differ from those from BM , may suggest an organ-specific activity of lung MSCs in controlling local tissue diseases [49, 50].
Finally, the in vivo search of SR4987PTX in metastatic lung demonstrated the presence of many Sca-1+ cells in parenchyma and even inside metastatic nodules. The few Sca-1+ cells present in the lung of control and PTX treated mice suggested a modest endogenous mobilization of MSCs from BM, at least at the time of mice sacrifice. Notably, we observed that Sca-1+ cells in mice injected with SR4987PTX were mostly detected nearby metastases or even attached to endothelium of the microvessels infiltrating metastatic foci; thus very close to cancer cells. To note that the vicinity of Dr-MCs to cancer cells is an important condition to ensure the efficacy of their toxic cargo (PTX) [15, 16]. In our opinion this result could explain the potent anti-metastatic effect obtained even when mice were treated with a lower dose of SR4987PTX.
Tumor recruits MSCs from host BM to form its stroma [41, 42, 51] a mechanism probably essential for tumor progression [45–48]. We can use this cancer property to fight it by administrating exogenous MSCs that can work as Trojan horses carrying in the tumor anticancer molecules . Although the presence of some conflicting data [52, 53], in general preclinical studies indicate that MSCs loaded with anti-cancer molecules could be very effective to inhibit primary and metastatic cancer growth [3–6]. The results shown here clearly demonstrated that our approach to upload anticancer drug, such as PTX, on MSCs lead them to become strongly cytotoxic against cancer and, importantly, without altering their home properties. 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.
This work was partially supported by a grant from Italian Association for Cancer Research # 14337 to C. Leonetti. We thank Franco Alpi and Lucia Fontana for their technical assistance. We thank Rosalindt Hendrix for checking the English of the manuscript.
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