Author’s Accepted Manuscript Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b+ myeloid cells to the lungs and facilitates B16-F10 melanoma colonization Lucas EB Souza, Danilo C Almeida, Juliana NU Yaochite, Dimas T Covas, Aparecida M Fontes
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To appear in: Experimental Cell Research Received date: 21 February 2015 Revised date: 25 May 2015 Accepted date: 26 May 2015 Cite this article as: Lucas EB Souza, Danilo C Almeida, Juliana NU Yaochite, Dimas T Covas and Aparecida M Fontes, Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b+ myeloid cells to the lungs and facilitates B16-F10 melanoma c o l o n i z a t i o n , Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2015.05.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b+ myeloid cells to the lungs and facilitates B16-F10 melanoma colonization Lucas EB Souza1,2, Danilo C Almeida4, Juliana NU Yaochite5, Dimas T Covas1,2, Aparecida M Fontes3§. 1
Department of Clinical Medicine, School of Medicine of Ribeirão Preto, University of São Paulo,
Ribeirão Preto, SP, Brazil. 2
Hemotherapy Center of Ribeirão Preto, School of Medicine of Ribeirão Preto, University of São
Paulo, Ribeirão Preto, SP, Brazil. 3
Department of Genetics, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão
Preto, SP, Brazil. 4
Department of Medicine – Nephrology, Laboratory of Clinical and Experimental Immunology,
Federal University of São Paulo, São Paulo, SP, Brazil. 5
Department of Biochemistry and Immunology, Basic and Applied Immunology Program, School of
Medicine of Ribeirão Preto, University of São Paulo, Brazil. §
Corresponding author
Email addresses: LEBS: [email protected] DCA: [email protected] JNUY: [email protected] DTC: [email protected] AMF: [email protected]
Abstract The discovery that the regenerative properties of bone marrow multipotent mesenchymal stromal cells (BM-MSCs) could collaterally favor neoplastic progression has led to a great interest in the function of these cells in tumors. However, the effect of BM-MSCs on colonization, a rate-limiting
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step of the metastatic cascade, is unknown. In this study, we investigated the effect of BM-MSCs on metastatic outgrowth of B16-F10 melanoma cells. In in vitro experiments, direct co-culture assays demonstrated that BM-MSCs stimulated the proliferation of B16-F10 cells in a dose-dependent manner. For in vivo experiments, luciferase-expressing B16-F10 cells were injected through tail vein and mice were subsequently treated with four systemic injections of BM-MSCs. In vivo bioluminescent imaging during 16 days demonstrated that BM-MSCs enhanced the colonization of lungs by B16-F10 cells, which correlated with a 2-fold increase in the number of metastatic foci. Flow cytometry analysis of lungs demonstrated that although mice harboring B16-F10 metastases displayed more endothelial cells, CD4 T and CD8 T lymphocytes in the lungs in comparison to metastases-free mice, BM-MSCs did not alter the number of these cells. Interestingly, BM-MSCs inoculation resulted in a 2-fold increase in the number of CD11b+ myeloid cells in the lungs of melanoma-bearing animals, a cell population previously described to organize “premetastatic niches” in experimental models. These findings indicate that BM-MSCs provide support to B16-F10 cells to overcome the constraints that limit metastatic outgrowth and that these effects might involve the interplay between BM-MSCs, CD11b+ myeloid cells and tumor cells.
1. Introduction Bone marrow-derived multipotent mesenchymal stromal cells (BM-MSCs) are perivascular progenitor cells that compose a hematopoietic niche where they provide support to hematopoiesis [1]. These cells are capable of osteogenic, adipogenic and chondrogenic differentiation in vitro [2] and form ectopic ossicles containing osteocytes, adipocytes and marrow stroma upon transplantation in mice [3]. After ex vivo expansion, cultured BM-MSCs display a wide repertoire of regenerative properties, which has broadened their clinical application. Among these capabilities, BM-MSCs are able to modulate the proliferation and activity of virtually all cells from immune system, to stimulate angiogenesis and to protect other cells (e.g. hepatocytes) from apoptosis [4]. Furthermore, it has been
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postulated that BM-MSCs possess tropism to injury sites and tumors [5], which have justified their use as cellular vehicles for directly delivering anti-cancer drugs into tumors [6]. Despite its complexity, the neoplastic disease is characterized by a limited set of biological capabilities collectively known as the hallmarks of cancer. They include induction of angiogenesis, evasion of growth suppression and circumvention of immune destruction [7]. Due to their regenerative properties, BM-MSCs are potentially capable of supporting most of these hallmarks. In addition, MSCs are natural components of tumor microenvironment since they were already isolated from several samples of human malignancies [8–11]. These considerations have fueled research efforts to unravel the role of BM-MSCs on tumor progression not only to shed light on the biology of cancer but also to develop BM-MSCs as a safe therapeutic tool. As reviewed elsewhere [12], numerous studies have addressed the role of BM-MSCs on tumor progression and most of which are based on the co-injection of BM-MSCs and tumor cells. Although very informative, this strategy does not recapitulate the current clinical setting, in which BM-MSCs are mainly administered systemically and thus can interact with tumor cells at colonization sites were the microenvironmental constraints are distinct from those found at primary tumors [13]. Furthermore, contrasting the extensive information about the role of BM-MSCs on primary tumor growth, little is known about the effect of these cells on metastasis, the main cause of death in patients with cancer. Clinical and experimental studies have been long demonstrating that metastasis is an inefficient process in which post-colonization growth constitutes one of the most important ratelimiting steps [14]. Early studies using mouse melanoma B16 cells showed that although nearly 80% of systemically injected cells survive and extravasate to lungs or liver parenchyma, only about 0.05% will form macroscopic metastases, while many individual tumor cells or micrometastases remain dormant [15, 16]. The mechanisms limiting the metastatic outgrowth are still poorly known, but activation of the angiogenic switch, activation of stem cell pathways such as Wnt and Notch by
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circumjacent stromal cells and suppression of immunological surveillance were already shown to stimulate the growth of dormant cancer cells into macrometastases [17]. Since BM-MSCs were described to exert therapeutic benefits through many of these mechanisms [4], we hypothesized that they could collaterally favor tumor colonization. Elucidating the effect of BM-MSCs on metastatic outgrowth would be important to not only because they have been explored as drug-delivery vehicles to tumors but also because several clinical evidences indicate the presence of dormant metastatic tumor cells in patients [17]. In this study, we investigated the effect of serial intravenous injections of BM-MSCs on the metastatic outgrowth during experimental lung colonization of B16-F10 melanoma cells. To shed light on the putative mechanisms involved in this process, we evaluated the mitogenic effects of BM-MSCs on B16-F10 cells in co-culture assays and also quantified populations of endothelial cells, CD4 T lymphocytes, CD8 T lymphocytes and myeloid cells in the lungs during metastatic outgrowth.
2. Methods 2.1. Animals
Animals used in this study were female, C57BL/6J mice, 8-12 weeks old. All procedures were done in accordance with institutional guidelines under protocols approved by the Ethics Comission for Animal Experiments from Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil.
2.2. Cells
Primary BM-MSCs were isolated from bone marrow of tibiae and femurs of C57BL/6J mice. After flushing out the bone marrow, obtained cells were washed twice in phosphate buffered solution (PBS), and plated at 2 x 106 cells/cm2 in minimal alpha essential medium (αMEM, Gibco)
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supplemented with 15% of fetal bovine serum (FBS), 26 mM sodium bicarbonate, 10 mM HEPES, 100 U/mL penicillin, and 100 µM streptomycin. After 72 hours, non-adherent cells were discarded by removing the medium and fresh medium was added. Adherent cells were subcultured at ~6x103 cells/cm2 and used for experiments after the 7th passage, when the cell population was free of hematopoietic (CD45+) and endothelial (CD31+) cells. Luciferase-expressing B16-F10 mouse melanoma cells (referred as B16luc cells) were purchased from Perkin Elmer and cultured in RPMI medium (Life technologies) supplemented with 10% FBS (Hyclone), 26mM sodium bicarbonate, 10mM HEPES, 100 U/mL penicillin, and 100 µM streptomycin.
2.3. Characterization of BM-MSCs
Suspensions containing 5x105 BM-MSCs in 50 μL of PBS were incubated with 0.5 μg of PEconjugated monoclonal antibodies anti-CD11b, anti-CD31, anti-CD34, anti-CD44, anti-CD45, antiCD90.2 and anti-Sca-1 (BD Pharmingen) during 15 min in the dark at room temperature (RT). Cells were then washed once in PBS and immediately submitted to flow cytometry analysis. The adipogenic and osteogenic potential of BM-MSCs were tested after culture in specific differentiation inductive medium as previously described [18]. Briefly, for adipogenic differentiation, BM-MSCs were cultured in αMEM supplemented with 15% FBS, 1 μM dexamethasone, 10 μg/mL insulin and 100 μM indomethacin. For osteogenic differentiation, BMMSCs were cultured in αMEM supplemented with 7,5% FBS, 0.1 μM dexamethasone, 200 μM ascorbic acid and 10 mM β-glycerophosphate. BM-MSCs were maintained in differentiation inductive media for 21 days. After this period, cells were fixed in 4% paraformaldehyde and stained with Sudan II for detection of lipid droplets or by the von Kossa method for identification of mineralized extracellular matrix. The samples were counter-stained with Harris’ Hematoxylin and photographed.
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2.4. Bioluminescent imaging (BLI)
BLI was performed using the Lumina In Vivo Imaging System (IVIS Lumina, Perkin Elmer). For in vitro imaging, D-luciferin (Perkin Elmer) was added to each well at a final concentration of 150 µg/ml 1-2 min prior to imaging. For in vivo imaging, mice were intraperitoneally injected with 150 mg/kg of D-luciferin and placed into the IVIS light-tight camera box under continuous exposure to 1.5% isoflurane. The exposure time ranged from 10 s to 5 min, depending on the bioluminescence intensity. For bioluminescence quantification, a region of interest (ROI) was manually drawn to encompass the bioluminescent signal and the intensity was recorded as photons/s.
2.5. Experimental metastasis model
In order to evaluate the effects of BM-MSCs on B16luc experimental metastases, C57BL/6J mice (n = 10) were first injected with 3x105 B16luc cells in 150 μL PBS through caudal vein. The day of infusion of B16luc cells was marked as D+0. At D+2 and D+6, half of the mice received intravenous injections of 6x105 BM-MSCs. At D+10 and D+14, these mice received additional injections of 3x105 BM-MSCs. Additional control animals (n = 3) received only BM-MSCs or PBS intravenously. Mice receiving B16luc cells were submitted to in vivo BLI at the time points D+4, D+8, D+12 and D+16 in order to monitor the outgrowth of melanoma metastases. At D+17, lungs were surgically removed, inflated by endotracheal infusion of PBS and photographed. The number of macroscopic melanoma metastases in the lungs was determined under a dissecting microscope. After the documentation, lungs were digested in collagenase IA for quantification of endothelial cells, macrophages and lymphocytes by flow cytometry.
2.6. Quantification of cell populations in the lungs
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Whole excised lungs were cleared from upper airways (trachea and bronchius) and finely minced with razor blades. Next, each sample were incubated in 3 mL of 0.1% collagenase IA (Sigma-Aldrich) dissolved in RPMI medium (Life Techonologies) without FBS at 37 °C for 40 min under agitation at every 10 min. Digestion was interrupted by adding 3 mL of RPMI medium supplemented with 10% FBS. Samples were filtered through 0.45 μm cell strainers followed by erythrocytes lysis by incubation in ACK buffer for 5 min at RT. The obtained cell suspension were counted and incubated with of the following monoclonal antibodies (1 μg/106 cells): PE-conjugated anti-CD31 for quantification of CD31+ endothelial cells; PE-conjugated anti-CD11b for detection of CD11b+ myeloid cells; FITC-conjugated anti-CD3, PerCP-conjugated anti-CD4 and APCconjugated anti-CD8 for quantification of CD4 T lymphocytes (CD3+CD4+) or CD8 T lymphocytes (CD3+CD8+).
2.7. Co-culture of B16luc cells and MSCs
Initially, 103, 2x103, 4x103 and 8x103 BM-MSCs were seeded in triplicates in a black 24 well plate and cultured for 24 h. Next, each well received 2x103 B16luc cells, resulting in the following ratio between melanoma cells and BM-MSCs: 2:1, 1:1, 1:2 and 1:4 ratio. B16luc cells cultured alone served as a control. After plating all cells, the growth of B16luc cells was daily monitored by in vitro bioluminescent imaging (BLI) during 5 days, after when the cells formed a confluent monolayer.
2.8. Statistical analysis
Numerical data are presented as mean ± standard error. Linear regression was performed to model the linear relationship between two variables. Student’s t test or analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test was carried out to compare means. Statistical analysis was done with Graphpad prism 5.0 software and the level of significance was fixed at p < 0.05. 7
3. Results 3.1. Characterization of bone marrow-derived mesenchymal stromal cells
BM-MSCs were isolated by culturing the adherent fraction of whole bone marrow from tibiae and femurs of C57BL/6J mice (n = 4). After three passages in culture, the population of adherent bone marrow cells was composed by morphologically distinct cell types (figure 1A, left panel) and contained many endothelial (CD31+) and hematopoietic (CD11b+, CD45+, CD34+) cell contaminants, as verified by analysis of cell surface markers by flow cytometry (figure 1B, red bars). However, at the sixth passage we obtained a morphologically homogeneous population of fibroblastic BM-MSCs (figure 1A, right panel), which were free of hematopoietic and endothelial cell contaminants (figure 1B, blue bars). We also confirmed that these BM-MSCs were capable of differentiating in vitro towards adipogenic and osteogenic lineages (figures 1C and 1D). Therefore, BM-MSCs were considered suitable for transplantation after 6 passages in culture, when they displayed their typical immunophenotypic profile and in vitro multilineage differentiation capacity.
3.2. Serial administration of BM-MSCs facilitates lung colonization by B16-F10 melanoma cells
The hypothesis of this work is that systemic administration of BM-MSCs facilitates the colonization of lungs by B16-F10 melanoma cells. In order to monitor the formation of metastases non-invasively, we used B16-F10 cells expressing the bioluminescent reporter firefly luciferase (B16luc cells). As verified by in vitro BLI, the bioluminescent signal was linearly and directly proportional to the number of B16luc cells (R2 = 0.99, figure 2A). For the establishment of lung metastases, C57BL/6J mice were intravenously injected with 3x105 B16luc cells through the caudal vein. Next, animals received single intravenous injections of 6x105 MSCs at days 2 and 6 after tumor cell inoculation. Another two injections of MSCs were
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given at days 10 and 14, however, the burden of lung metastases lead us to reduce the amount of administered BM-MSCs to 3x105 cells in order to avoid animal death by pulmonary embolism. The bioluminescent signal from injected B16luc cells was regularly monitored by in vivo BLI. Sixteen days after tumor cell inoculation, mice receiving serial injections of BM-MSCs displayed a two-fold higher bioluminescent signal in the thorax than the animals injected with B16luc cells alone (3.68x106 ± 1.3x106 vs. 7.28x106 ± 1.91x106 photons/s, p = 0.03, figures 2B and 2C). Subsequent macroscopic analysis of the lungs also showed that the number of superficial macrometastases was 42% higher in animals treated with BM-MSCs in comparison to mice receiving B16luc cells alone (194.4 ± 11.4 vs. 277.8 ± 15.8 metastases, p < 0.01, figures 2D and 2E).
3.3. BM-MSCs stimulate the proliferation of B16 melanoma cells in direct co-cultures
Cultured BM-MSCs are known to stimulate the proliferation of distinct cells, which could have contributed to the facilitation of B16luc metastatic outgrowth. Thus, we investigate whether this mitogenic property of BM-MSCs is recapitulated during the interaction with B16luc cells. To accomplish this, we co-cultured BM-MSCs and B16luc at different proportions and evaluated the proliferation of B16luc cells using in vitro BLI. Continuous monitoring of B16luc cells bioluminescence for 5 days demonstrated that the addition of BM-MSCs in all tested proportions significantly accelerated the proliferation of melanoma cells in vitro (figures 3A and 3B). We next estimated the velocity of growth of B16luc cells by calculating the area under curve of the graphs obtained from bioluminescence quantification. As a result, we found that the area under curve was directly proportional to the amount of BM-MSCs added to B16luc cell cultures (figure 3C). These data demonstrate that BM-MSCs directly stimulate the proliferation of B16luc melanoma cells in a dose-dependent manner.
3.4. Serial administration of BM-MSCs increases the number of CD11b+ myeloid cells in the lungs of melanoma-bearing mice
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Inflammatory cells that compose the tumor microenvironment are known to play a pivotal role in either primary tumor or metastases growth. Thus, we tested whether serial inoculation of BMMSCs during B16luc metastatic outgrowth would modulate the number of CD11b+ myeloid cells (MCs) in the lungs. After the complete digestion of the lungs with collagenase, flow cytometry analysis demonstrated that the development of lung metastases in mice receiving only B16luc cells was accompanied by a 3-fold increase in total number of CD11b+ MCs in comparison to metastases-free mice that received only PBS (5.11x105 ± 2.11x105 vs. 1.53x106 ± 3.47x105 CD11b+ cells, p = 0.03, figure 4). Notably, treatment of mice with intravenous injections of BM-MSCs further enhanced the number of CD11b+ MCs in the lungs in comparison to mice receiving B16luc cells alone (1.53x106 ± 3.47x105 vs. 2.94x106 ± 5.80x105 CD11b+ cells, p = 0.04, figure 4).
3.5. Administration of BM-MSCs does not alter the number of endothelial cells or lymphocytes in lungs with melanoma metastases
As part of their regenerative repertoire, BM-MSCs are known for their pro-angiogenic and immunosuppressive properties, which in turn could collaterally support the metastatic colonization if the growth of metastases is being limited by the lack of adequate blood supply or by an ongoing immunological response. We therefore aimed to quantify the populations of CD31+ endothelial cells and both CD3+CD8+ and CD3+CD4+ T lymphocytes in the lungs after treatment with BM-MSCs. Histological analysis of the lungs demonstrated the presence of blood vessels within melanoma macrometastases (figure 5A), indicating the activation of angiogenesis at these sites. Accordingly, mice with lung melanoma metastases displayed more than twice CD31+ endothelial cells in the lungs in comparison to metastases-free mice, as evaluated by flow cytometry (1.94x106 ± 6.29x105 vs. 4.62x106 ± 3.85x105 CD31+ cells, p = 0.02, figure 5B-C). However, treatment of mice
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with serial inoculations of BM-MSCs showed no effect on the number of lung CD31+ endothelial cells (4.62x106 ± 3.85x105 vs. 5.37x106 ± 9.59x105 CD31+, p = 0.50, figure 5B-C). The colonization of the lungs by B16luc melanoma cells also correlated with an increase in the number of both CD4 T and CD8 T lymphocytes in comparison to metastases-free mice (CD4+ cells: 3.61x105 ± 1.40x105 vs. 1.44x106 ± 1.74x105 cells, p < 0,01; CD8+ cells: 1.45x105 ± 5.09x104 vs. 7.83x105 ± 8.86x104 cells, p < 0.01, figure 6). However, despite the well-described immunosuppressive properties of BM-MSCs, we found that they did not reduce the number of either CD4 T or CD8 T lymphocytes in the lungs of melanoma-bearing mice (CD4+ cells: 1.44x106 ± 1.74x105 vs. 2.00x106 ± 2.04x105 cells, p = 0,08; CD8+ cells: 7.83x105 ± 6.34x104 vs. 9.20x105 ± 6.34x104 cells, p = 0.25, figure 6).
4. Discussion and Conclusion The formation of metastatic nodules after tumor cell extravasation (i.e. colonization) is a ratelimiting event of the metastatic cascade. Although primary tumors shed millions of cells daily in the blood stream [19–21], formation of metastases is rare, partly due to the absence of growthsupporting signals at colonization sites. Here, we demonstrated that intravenously administered BMMSCs enhanced the colonization capacity of B16-F10 cells, thereby increasing the incidence of lung metastases. In this study, we adopted a serial intravenous administration of BM-MSCs in order to recapitulate a delivery strategy commonly used for treatment of systemic diseases in clinical trials [22] and also used to enhance cell engraftment in pre-clinical models [23]. After BM-MSCs inoculation in mice previously injected with melanoma cells, the tumor burden in the lungs increased, as well as the number of superficial macrometastases, demonstrating that BM-MSCs enhanced the colonization capacity of B16-F10 melanoma cells. These results are in line with the
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findings of Shinagawa et al., who observed that intrasplenic co-injection of KM12SM colon cancer cells and MSCs leads to the formation of more liver metastases than KM12SM injected alone [24]. On the other hand, Studeny et al. observed that systemic inoculation of human MSCs had no effect on the growth of experimental human breast cancer metastases in the lungs [25]. These discrepancies can be in part attributed to differences on experimental conditions like the use of distinct tumor cell types [12] or the use of immunodeficient mouse models that cannot recapitulate the interaction between MSCs and immune cells, which in turn may have a fundamental role during tumor progression. Of note, the supportive role of BM-MSCs during metastases formation observed in this study is in agreement with previous evidences demonstrating that colonization is not a cell-autonomous process but is rather dependent on the interaction between tumor and stromal cells at colonization sites. For instance, depletion of S100A4+ fibroblasts reduces the metastatic colonization without affecting the growth of primary mammary tumors in mice [26]. In addition, induction of periostin expression in lung stromal cells is required for the establishment of experimental breast cancer metastases in these organs [27]. Although we did not assessed in situ extravasation, it was previously reported that 80% of B16-F10 cells survive and extravasate within 24 h after injection into circulation [28]. Since the first administration of BM-MSCs was given 48 h after B16luc cells inoculation, their effect on colonization is likely to be a consequence of pro-mitogenic/survival stimuli rather than enhancement of extravasation. Accordingly, our in vivo BLI analysis revealed that the number of B16luc cells in the lungs during 8 days following injection was not altered by BM-MSCs administration. In order to shed light on the putative mechanisms underlying the promotion of B16-F10 metastatic outgrowth by BM-MSCs, we investigated whether some potentially pro-tumorigenic properties of BM-MSCs were activated during interaction with B16-F10 cells in vitro or during metastatic colonization in vivo.
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MSCs exert their therapeutic benefits through multiple mechanisms. One of them is through support to the growth of endogenous progenitor cells such as cardiac stem cells [29] and skeletal myoblasts [30]. The mitogenic stimuli provided by MSCs are mediated by secreted molecules such as the vascular endothelial growth factor (VEGF), or by direct cell-to-cell contact interactions [31]. Using direct co-culture assays, we found that BM-MSCs also stimulate the proliferation of B16-F10 melanoma cells in vitro. Moreover, the growth of B16-F10 cells was directly proportional to the number of BM-MSCs seeded in the co-culture assays. Although a clear demonstration of the mitogenic effect of BM-MSCs in vivo is challenging and remains to be done, our observations indicate that BM-MSCs are potentially capable of directly supporting the growth of B16-F10 cells during colonization. Numerous studies reported opposite effects of MSCs on tumor cell proliferation. As examples, human BM-MSCs secrete IL-6 which in turn activate STAT3 in the human osteosarcoma cell line Saos-2 and promote their growth in vitro [32]. Recently, it was demonstrated that MSCs derived from gastric cancer enhance the proliferation of gastric tumor cells through exosomal transference of microRNAs [33]. Conversely, MSCs were reported to suppress the proliferation of K562 leukemia cells through secretion of dickkopf-1 (DKK-1), a negative regulator of the WNT pathway [34]. Therefore, the effect of MSCs on tumor cell proliferation depends on which molecular circuitries are activated or repressed during this heterotypic interaction. As additional properties of their regenerative repertoire, MSCs are known to stimulate angiogenesis and to suppress the activation and proliferation of virtually all cells from immune system, which has broadened their potential clinical application to include the treatment of ischemic and immune-related diseases. However, these MSC properties were also responsible to promote human colorectal tumor growth through induction of angiogenesis [35] and to allow the development of melanoma after transplantation of B16-F10 cells in allogenic recipients through suppression of cytotoxic CD8+ T cells [36]. In the present study, administration of BM-MSCs did not alter the
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number of CD31+ endothelial cells or CD8+ and CD4+ T lymphocytes within metastases-bearing lungs. This indicates that facilitation of B16-F10 metastatic outgrowth was not mediated by induction of angiogenesis or suppression of immunological surveillance. Indeed, the poor immunogenicity of B16-F10 melanoma cells have been long known [37], thus it is expected that immunosuppression of the host is not critical for the metastatic colonization in our model. Tumors have been long conceived as wounds that never heal [38] and they are infiltrated by a myriad of immune cells, stromal cells and endothelial cells that compose the tumor stroma. Infiltrating CD11b+ myeloid cells, which comprise macrophages, neutrophils and monocytes, for example, have been shown to facilitate the acquisition of many hallmarks of cancer, including sustained proliferation, angiogenesis, invasion and metastasis [39, 40]. It was demonstrated that MSCs isolated from spontaneous mouse lymphoma enhances the recruitment of CD11b+ macrophages/monocytes to primary tumors via secretion of CCR2 ligands and that these infiltrating cells accelerate tumor growth [41]. In addition, studies using xenograft or spontaneous models of breast cancer demonstrated that CD11b+ myeloid cells are recruited to specific organs such as the lungs where they form a “premetastatic niche” and facilitate the outgrowth of metastatic tumor cells by promoting vascular remodeling through secretion of metalloproteinases [42] or by stimulating mesenchymal-to-epithelial transition and tumor cell proliferation through deposition of extracellular matrix components such as the proteoglycan versican [43]. In the present study, we demonstrated that systemically administered BM-MSCs enhanced the recruitment of CD11b+ myeloid cells to the lungs with developing melanoma metastases, suggesting an interplay between these cell populations during the promotion of B16-F10 metastatic outgrowth. Infiltrating CD11b+ myeloid cells encompass a broad spectrum of subpopulations whose contributions to the hallmarks of cancer have been extensively uncovered [39]. For instance, CD11b+Gr1+ myeloid cells and tumor-associated macrophages were described to promote tumor neovascularization through release of soluble mediators such as the vascular endothelial growth
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factor (VEGF) [44, 45]. In addition, distinct subsets of CD11b+Gr1+ cells display strong immunosuppressive properties which can favor tumor progression by impairing tumor-specific immune response [46, 47]. Despite these well-described functions, we observed that the higher recruitment of CD11b+ cells to the lungs after MSCs injection did not implicate in changes on the number of endothelial cells and T cell subsets. These correlative evidences indicate that the immunosuppressive and angiogenic functions of CD11b+ myeloid cells were not crucial for their support to metastatic outgrowth in our model. However, alternative mechanistic explanations remain possible such as the release of growth factors after proteolytic cleavage of extracellular matrix by CD11b+ cells-derived proteases [48] and protection against death of tumor cells triggered by integrin signaling during direct heterotypic interaction at colonization sites [49]. Due to their wide repertoire of regenerative properties, MSCs have been investigated to treat an equally broad range of local and systemic diseases such as ischemia, autoimmune diseases and transplant-related complications like the graft-versus-host disease [4]. Of note, MSCs also have a tropism to inflammation sites and tumors [5]. This particular characteristic has driven the use of genetically engineered MSCs to deliver anti-cancer molecules directly to developing tumors [6]. However, our findings demonstrate that systemically administered unmodified BM-MSCs might support the growth of metastases. These are valuable information for the progressive development of BM-MSCs as a safe therapeutic tool and imply that a thorough clinical inspection for the presence of incipient metastases should be carried out in patients before and during the therapy with systemically administered BM-MSCs. Furthermore, if applicable to other tumor types, our observations indicate that the efficiency of the metastatic colonization is affected by the interaction among BM-MSCs, myeloid cells and tumor cells at the target organs.
5. Competing interests The authors have no conflict of interests to declare.
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6. Acknowledgements The authors would like to thank Patrícia V. B. Palma and Camila Menezes for their support with flow citometry analysis. This work was funded with resources from São Paulo Research Foundation (FAPESP – Proc. 2008/08944-0), Coordination for Improvement of Higher Education Personnel (CAPES) and from the National Counsel of Technological and Scientific Development (CNPq Proc. 310619/2012-2).
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8. Figure legends Figure 1: Characterization of bone marrow-derived mesenchymal stromal cells (BM-MSCs). (A) Phase-contrast photomicrographs of BM-MSCs cultures at 3rd and 6th passage. At third passage, BMMSCs cultures were highly heterogeneous, as noticed by the presence of morphologically distinct cell types (left panel). At sixth passage, however, the cultures became morphologically homogeneous and cells displayed multipolar fibroblastic morphology, as expected for BM-MSCs (right panel). Magnification = 100x. (B) Expression of cell surface markers in BM-MSCs cultures evaluated by flow cytometry. At the third passage, BM-MSCs cultures were still contaminated with endothelial (CD31+ cells) and hematopoietic cells (CD11b+, CD45+ and CD34+ cells). However, after six passages in culture, these hematopoietic and endothelial cell contaminants were no longer present. As expected, BM-MSCs did not express the endothelial and hematopoietic markers CD11b, CD31, CD34 and CD45 but were positive for CD44, CD90.2 and Sca-1 (n = 3 independent cultures). (C) Adipogenic differentiation of BM-MSCs. After culture in adipogenic medium for 21 days, BMMSCs accumulated intracellular lipid vesicles, which were stained in orange after staining with Sudan II (left panel). Non-stimulated BM-MSCs did not undergo adipogenic differentiation (right panel). Magnification = 400x. (D) Osteogenic differentiation of BM-MSCs. After culture in osteogenic medium for 21 days, BM-MSCs synthesized mineralized extracellular matrix, which was stained in black or dark brown after staining by von Kossa’s method (left panel,). Cells maintained in regular medium did not undergo osteogenic differentiation (right panel). Magnification = 40x. Figure 2: Systemic inoculation of BM-MSCs facilitates lung colonization by B16luc melanoma cells. (A) Biological activity of luciferase in B16luc melanoma cells. The bioluminescent signal was directly proportional to the number of B16luc cells (linear regression test, R2 = 0.99). (B) Representative images of mice submitted to in vivo bioluminescent imaging to monitor the establishment and growth of melanoma metastases. Systemic administration of BM-MSCs enhanced 20
the lung colonization by B16luc cells (lower pictures) in comparison to mice receiving B16luc cells alone. (C) Quantification of bioluminescent signal from thoracic region of melanoma-bearing mice. Sixteen days after B16luc cells injection, mice treated with BM-MSCs displayed a higher lung metastases burden (blue line) in comparison to mice receiving B16luc cells alone (n = 5 animals per group; *p
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S0014-4827(15)30005-7 http://dx.doi.org/10.1016/j.yexcr.2015.05.021 YEXCR9962
To appear in: Experimental Cell Research Received date: 21 February 2015 Revised date: 25 May 2015 Accepted date: 26 May 2015 Cite this article as: Lucas EB Souza, Danilo C Almeida, Juliana NU Yaochite, Dimas T Covas and Aparecida M Fontes, Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b+ myeloid cells to the lungs and facilitates B16-F10 melanoma c o l o n i z a t i o n , Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2015.05.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b+ myeloid cells to the lungs and facilitates B16-F10 melanoma colonization Lucas EB Souza1,2, Danilo C Almeida4, Juliana NU Yaochite5, Dimas T Covas1,2, Aparecida M Fontes3§. 1
Department of Clinical Medicine, School of Medicine of Ribeirão Preto, University of São Paulo,
Ribeirão Preto, SP, Brazil. 2
Hemotherapy Center of Ribeirão Preto, School of Medicine of Ribeirão Preto, University of São
Paulo, Ribeirão Preto, SP, Brazil. 3
Department of Genetics, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão
Preto, SP, Brazil. 4
Department of Medicine – Nephrology, Laboratory of Clinical and Experimental Immunology,
Federal University of São Paulo, São Paulo, SP, Brazil. 5
Department of Biochemistry and Immunology, Basic and Applied Immunology Program, School of
Medicine of Ribeirão Preto, University of São Paulo, Brazil. §
Corresponding author
Email addresses: LEBS: [email protected] DCA: [email protected] JNUY: [email protected] DTC: [email protected] AMF: [email protected]
Abstract The discovery that the regenerative properties of bone marrow multipotent mesenchymal stromal cells (BM-MSCs) could collaterally favor neoplastic progression has led to a great interest in the function of these cells in tumors. However, the effect of BM-MSCs on colonization, a rate-limiting
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step of the metastatic cascade, is unknown. In this study, we investigated the effect of BM-MSCs on metastatic outgrowth of B16-F10 melanoma cells. In in vitro experiments, direct co-culture assays demonstrated that BM-MSCs stimulated the proliferation of B16-F10 cells in a dose-dependent manner. For in vivo experiments, luciferase-expressing B16-F10 cells were injected through tail vein and mice were subsequently treated with four systemic injections of BM-MSCs. In vivo bioluminescent imaging during 16 days demonstrated that BM-MSCs enhanced the colonization of lungs by B16-F10 cells, which correlated with a 2-fold increase in the number of metastatic foci. Flow cytometry analysis of lungs demonstrated that although mice harboring B16-F10 metastases displayed more endothelial cells, CD4 T and CD8 T lymphocytes in the lungs in comparison to metastases-free mice, BM-MSCs did not alter the number of these cells. Interestingly, BM-MSCs inoculation resulted in a 2-fold increase in the number of CD11b+ myeloid cells in the lungs of melanoma-bearing animals, a cell population previously described to organize “premetastatic niches” in experimental models. These findings indicate that BM-MSCs provide support to B16-F10 cells to overcome the constraints that limit metastatic outgrowth and that these effects might involve the interplay between BM-MSCs, CD11b+ myeloid cells and tumor cells.
1. Introduction Bone marrow-derived multipotent mesenchymal stromal cells (BM-MSCs) are perivascular progenitor cells that compose a hematopoietic niche where they provide support to hematopoiesis [1]. These cells are capable of osteogenic, adipogenic and chondrogenic differentiation in vitro [2] and form ectopic ossicles containing osteocytes, adipocytes and marrow stroma upon transplantation in mice [3]. After ex vivo expansion, cultured BM-MSCs display a wide repertoire of regenerative properties, which has broadened their clinical application. Among these capabilities, BM-MSCs are able to modulate the proliferation and activity of virtually all cells from immune system, to stimulate angiogenesis and to protect other cells (e.g. hepatocytes) from apoptosis [4]. Furthermore, it has been
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postulated that BM-MSCs possess tropism to injury sites and tumors [5], which have justified their use as cellular vehicles for directly delivering anti-cancer drugs into tumors [6]. Despite its complexity, the neoplastic disease is characterized by a limited set of biological capabilities collectively known as the hallmarks of cancer. They include induction of angiogenesis, evasion of growth suppression and circumvention of immune destruction [7]. Due to their regenerative properties, BM-MSCs are potentially capable of supporting most of these hallmarks. In addition, MSCs are natural components of tumor microenvironment since they were already isolated from several samples of human malignancies [8–11]. These considerations have fueled research efforts to unravel the role of BM-MSCs on tumor progression not only to shed light on the biology of cancer but also to develop BM-MSCs as a safe therapeutic tool. As reviewed elsewhere [12], numerous studies have addressed the role of BM-MSCs on tumor progression and most of which are based on the co-injection of BM-MSCs and tumor cells. Although very informative, this strategy does not recapitulate the current clinical setting, in which BM-MSCs are mainly administered systemically and thus can interact with tumor cells at colonization sites were the microenvironmental constraints are distinct from those found at primary tumors [13]. Furthermore, contrasting the extensive information about the role of BM-MSCs on primary tumor growth, little is known about the effect of these cells on metastasis, the main cause of death in patients with cancer. Clinical and experimental studies have been long demonstrating that metastasis is an inefficient process in which post-colonization growth constitutes one of the most important ratelimiting steps [14]. Early studies using mouse melanoma B16 cells showed that although nearly 80% of systemically injected cells survive and extravasate to lungs or liver parenchyma, only about 0.05% will form macroscopic metastases, while many individual tumor cells or micrometastases remain dormant [15, 16]. The mechanisms limiting the metastatic outgrowth are still poorly known, but activation of the angiogenic switch, activation of stem cell pathways such as Wnt and Notch by
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circumjacent stromal cells and suppression of immunological surveillance were already shown to stimulate the growth of dormant cancer cells into macrometastases [17]. Since BM-MSCs were described to exert therapeutic benefits through many of these mechanisms [4], we hypothesized that they could collaterally favor tumor colonization. Elucidating the effect of BM-MSCs on metastatic outgrowth would be important to not only because they have been explored as drug-delivery vehicles to tumors but also because several clinical evidences indicate the presence of dormant metastatic tumor cells in patients [17]. In this study, we investigated the effect of serial intravenous injections of BM-MSCs on the metastatic outgrowth during experimental lung colonization of B16-F10 melanoma cells. To shed light on the putative mechanisms involved in this process, we evaluated the mitogenic effects of BM-MSCs on B16-F10 cells in co-culture assays and also quantified populations of endothelial cells, CD4 T lymphocytes, CD8 T lymphocytes and myeloid cells in the lungs during metastatic outgrowth.
2. Methods 2.1. Animals
Animals used in this study were female, C57BL/6J mice, 8-12 weeks old. All procedures were done in accordance with institutional guidelines under protocols approved by the Ethics Comission for Animal Experiments from Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil.
2.2. Cells
Primary BM-MSCs were isolated from bone marrow of tibiae and femurs of C57BL/6J mice. After flushing out the bone marrow, obtained cells were washed twice in phosphate buffered solution (PBS), and plated at 2 x 106 cells/cm2 in minimal alpha essential medium (αMEM, Gibco)
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supplemented with 15% of fetal bovine serum (FBS), 26 mM sodium bicarbonate, 10 mM HEPES, 100 U/mL penicillin, and 100 µM streptomycin. After 72 hours, non-adherent cells were discarded by removing the medium and fresh medium was added. Adherent cells were subcultured at ~6x103 cells/cm2 and used for experiments after the 7th passage, when the cell population was free of hematopoietic (CD45+) and endothelial (CD31+) cells. Luciferase-expressing B16-F10 mouse melanoma cells (referred as B16luc cells) were purchased from Perkin Elmer and cultured in RPMI medium (Life technologies) supplemented with 10% FBS (Hyclone), 26mM sodium bicarbonate, 10mM HEPES, 100 U/mL penicillin, and 100 µM streptomycin.
2.3. Characterization of BM-MSCs
Suspensions containing 5x105 BM-MSCs in 50 μL of PBS were incubated with 0.5 μg of PEconjugated monoclonal antibodies anti-CD11b, anti-CD31, anti-CD34, anti-CD44, anti-CD45, antiCD90.2 and anti-Sca-1 (BD Pharmingen) during 15 min in the dark at room temperature (RT). Cells were then washed once in PBS and immediately submitted to flow cytometry analysis. The adipogenic and osteogenic potential of BM-MSCs were tested after culture in specific differentiation inductive medium as previously described [18]. Briefly, for adipogenic differentiation, BM-MSCs were cultured in αMEM supplemented with 15% FBS, 1 μM dexamethasone, 10 μg/mL insulin and 100 μM indomethacin. For osteogenic differentiation, BMMSCs were cultured in αMEM supplemented with 7,5% FBS, 0.1 μM dexamethasone, 200 μM ascorbic acid and 10 mM β-glycerophosphate. BM-MSCs were maintained in differentiation inductive media for 21 days. After this period, cells were fixed in 4% paraformaldehyde and stained with Sudan II for detection of lipid droplets or by the von Kossa method for identification of mineralized extracellular matrix. The samples were counter-stained with Harris’ Hematoxylin and photographed.
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2.4. Bioluminescent imaging (BLI)
BLI was performed using the Lumina In Vivo Imaging System (IVIS Lumina, Perkin Elmer). For in vitro imaging, D-luciferin (Perkin Elmer) was added to each well at a final concentration of 150 µg/ml 1-2 min prior to imaging. For in vivo imaging, mice were intraperitoneally injected with 150 mg/kg of D-luciferin and placed into the IVIS light-tight camera box under continuous exposure to 1.5% isoflurane. The exposure time ranged from 10 s to 5 min, depending on the bioluminescence intensity. For bioluminescence quantification, a region of interest (ROI) was manually drawn to encompass the bioluminescent signal and the intensity was recorded as photons/s.
2.5. Experimental metastasis model
In order to evaluate the effects of BM-MSCs on B16luc experimental metastases, C57BL/6J mice (n = 10) were first injected with 3x105 B16luc cells in 150 μL PBS through caudal vein. The day of infusion of B16luc cells was marked as D+0. At D+2 and D+6, half of the mice received intravenous injections of 6x105 BM-MSCs. At D+10 and D+14, these mice received additional injections of 3x105 BM-MSCs. Additional control animals (n = 3) received only BM-MSCs or PBS intravenously. Mice receiving B16luc cells were submitted to in vivo BLI at the time points D+4, D+8, D+12 and D+16 in order to monitor the outgrowth of melanoma metastases. At D+17, lungs were surgically removed, inflated by endotracheal infusion of PBS and photographed. The number of macroscopic melanoma metastases in the lungs was determined under a dissecting microscope. After the documentation, lungs were digested in collagenase IA for quantification of endothelial cells, macrophages and lymphocytes by flow cytometry.
2.6. Quantification of cell populations in the lungs
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Whole excised lungs were cleared from upper airways (trachea and bronchius) and finely minced with razor blades. Next, each sample were incubated in 3 mL of 0.1% collagenase IA (Sigma-Aldrich) dissolved in RPMI medium (Life Techonologies) without FBS at 37 °C for 40 min under agitation at every 10 min. Digestion was interrupted by adding 3 mL of RPMI medium supplemented with 10% FBS. Samples were filtered through 0.45 μm cell strainers followed by erythrocytes lysis by incubation in ACK buffer for 5 min at RT. The obtained cell suspension were counted and incubated with of the following monoclonal antibodies (1 μg/106 cells): PE-conjugated anti-CD31 for quantification of CD31+ endothelial cells; PE-conjugated anti-CD11b for detection of CD11b+ myeloid cells; FITC-conjugated anti-CD3, PerCP-conjugated anti-CD4 and APCconjugated anti-CD8 for quantification of CD4 T lymphocytes (CD3+CD4+) or CD8 T lymphocytes (CD3+CD8+).
2.7. Co-culture of B16luc cells and MSCs
Initially, 103, 2x103, 4x103 and 8x103 BM-MSCs were seeded in triplicates in a black 24 well plate and cultured for 24 h. Next, each well received 2x103 B16luc cells, resulting in the following ratio between melanoma cells and BM-MSCs: 2:1, 1:1, 1:2 and 1:4 ratio. B16luc cells cultured alone served as a control. After plating all cells, the growth of B16luc cells was daily monitored by in vitro bioluminescent imaging (BLI) during 5 days, after when the cells formed a confluent monolayer.
2.8. Statistical analysis
Numerical data are presented as mean ± standard error. Linear regression was performed to model the linear relationship between two variables. Student’s t test or analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test was carried out to compare means. Statistical analysis was done with Graphpad prism 5.0 software and the level of significance was fixed at p < 0.05. 7
3. Results 3.1. Characterization of bone marrow-derived mesenchymal stromal cells
BM-MSCs were isolated by culturing the adherent fraction of whole bone marrow from tibiae and femurs of C57BL/6J mice (n = 4). After three passages in culture, the population of adherent bone marrow cells was composed by morphologically distinct cell types (figure 1A, left panel) and contained many endothelial (CD31+) and hematopoietic (CD11b+, CD45+, CD34+) cell contaminants, as verified by analysis of cell surface markers by flow cytometry (figure 1B, red bars). However, at the sixth passage we obtained a morphologically homogeneous population of fibroblastic BM-MSCs (figure 1A, right panel), which were free of hematopoietic and endothelial cell contaminants (figure 1B, blue bars). We also confirmed that these BM-MSCs were capable of differentiating in vitro towards adipogenic and osteogenic lineages (figures 1C and 1D). Therefore, BM-MSCs were considered suitable for transplantation after 6 passages in culture, when they displayed their typical immunophenotypic profile and in vitro multilineage differentiation capacity.
3.2. Serial administration of BM-MSCs facilitates lung colonization by B16-F10 melanoma cells
The hypothesis of this work is that systemic administration of BM-MSCs facilitates the colonization of lungs by B16-F10 melanoma cells. In order to monitor the formation of metastases non-invasively, we used B16-F10 cells expressing the bioluminescent reporter firefly luciferase (B16luc cells). As verified by in vitro BLI, the bioluminescent signal was linearly and directly proportional to the number of B16luc cells (R2 = 0.99, figure 2A). For the establishment of lung metastases, C57BL/6J mice were intravenously injected with 3x105 B16luc cells through the caudal vein. Next, animals received single intravenous injections of 6x105 MSCs at days 2 and 6 after tumor cell inoculation. Another two injections of MSCs were
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given at days 10 and 14, however, the burden of lung metastases lead us to reduce the amount of administered BM-MSCs to 3x105 cells in order to avoid animal death by pulmonary embolism. The bioluminescent signal from injected B16luc cells was regularly monitored by in vivo BLI. Sixteen days after tumor cell inoculation, mice receiving serial injections of BM-MSCs displayed a two-fold higher bioluminescent signal in the thorax than the animals injected with B16luc cells alone (3.68x106 ± 1.3x106 vs. 7.28x106 ± 1.91x106 photons/s, p = 0.03, figures 2B and 2C). Subsequent macroscopic analysis of the lungs also showed that the number of superficial macrometastases was 42% higher in animals treated with BM-MSCs in comparison to mice receiving B16luc cells alone (194.4 ± 11.4 vs. 277.8 ± 15.8 metastases, p < 0.01, figures 2D and 2E).
3.3. BM-MSCs stimulate the proliferation of B16 melanoma cells in direct co-cultures
Cultured BM-MSCs are known to stimulate the proliferation of distinct cells, which could have contributed to the facilitation of B16luc metastatic outgrowth. Thus, we investigate whether this mitogenic property of BM-MSCs is recapitulated during the interaction with B16luc cells. To accomplish this, we co-cultured BM-MSCs and B16luc at different proportions and evaluated the proliferation of B16luc cells using in vitro BLI. Continuous monitoring of B16luc cells bioluminescence for 5 days demonstrated that the addition of BM-MSCs in all tested proportions significantly accelerated the proliferation of melanoma cells in vitro (figures 3A and 3B). We next estimated the velocity of growth of B16luc cells by calculating the area under curve of the graphs obtained from bioluminescence quantification. As a result, we found that the area under curve was directly proportional to the amount of BM-MSCs added to B16luc cell cultures (figure 3C). These data demonstrate that BM-MSCs directly stimulate the proliferation of B16luc melanoma cells in a dose-dependent manner.
3.4. Serial administration of BM-MSCs increases the number of CD11b+ myeloid cells in the lungs of melanoma-bearing mice
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Inflammatory cells that compose the tumor microenvironment are known to play a pivotal role in either primary tumor or metastases growth. Thus, we tested whether serial inoculation of BMMSCs during B16luc metastatic outgrowth would modulate the number of CD11b+ myeloid cells (MCs) in the lungs. After the complete digestion of the lungs with collagenase, flow cytometry analysis demonstrated that the development of lung metastases in mice receiving only B16luc cells was accompanied by a 3-fold increase in total number of CD11b+ MCs in comparison to metastases-free mice that received only PBS (5.11x105 ± 2.11x105 vs. 1.53x106 ± 3.47x105 CD11b+ cells, p = 0.03, figure 4). Notably, treatment of mice with intravenous injections of BM-MSCs further enhanced the number of CD11b+ MCs in the lungs in comparison to mice receiving B16luc cells alone (1.53x106 ± 3.47x105 vs. 2.94x106 ± 5.80x105 CD11b+ cells, p = 0.04, figure 4).
3.5. Administration of BM-MSCs does not alter the number of endothelial cells or lymphocytes in lungs with melanoma metastases
As part of their regenerative repertoire, BM-MSCs are known for their pro-angiogenic and immunosuppressive properties, which in turn could collaterally support the metastatic colonization if the growth of metastases is being limited by the lack of adequate blood supply or by an ongoing immunological response. We therefore aimed to quantify the populations of CD31+ endothelial cells and both CD3+CD8+ and CD3+CD4+ T lymphocytes in the lungs after treatment with BM-MSCs. Histological analysis of the lungs demonstrated the presence of blood vessels within melanoma macrometastases (figure 5A), indicating the activation of angiogenesis at these sites. Accordingly, mice with lung melanoma metastases displayed more than twice CD31+ endothelial cells in the lungs in comparison to metastases-free mice, as evaluated by flow cytometry (1.94x106 ± 6.29x105 vs. 4.62x106 ± 3.85x105 CD31+ cells, p = 0.02, figure 5B-C). However, treatment of mice
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with serial inoculations of BM-MSCs showed no effect on the number of lung CD31+ endothelial cells (4.62x106 ± 3.85x105 vs. 5.37x106 ± 9.59x105 CD31+, p = 0.50, figure 5B-C). The colonization of the lungs by B16luc melanoma cells also correlated with an increase in the number of both CD4 T and CD8 T lymphocytes in comparison to metastases-free mice (CD4+ cells: 3.61x105 ± 1.40x105 vs. 1.44x106 ± 1.74x105 cells, p < 0,01; CD8+ cells: 1.45x105 ± 5.09x104 vs. 7.83x105 ± 8.86x104 cells, p < 0.01, figure 6). However, despite the well-described immunosuppressive properties of BM-MSCs, we found that they did not reduce the number of either CD4 T or CD8 T lymphocytes in the lungs of melanoma-bearing mice (CD4+ cells: 1.44x106 ± 1.74x105 vs. 2.00x106 ± 2.04x105 cells, p = 0,08; CD8+ cells: 7.83x105 ± 6.34x104 vs. 9.20x105 ± 6.34x104 cells, p = 0.25, figure 6).
4. Discussion and Conclusion The formation of metastatic nodules after tumor cell extravasation (i.e. colonization) is a ratelimiting event of the metastatic cascade. Although primary tumors shed millions of cells daily in the blood stream [19–21], formation of metastases is rare, partly due to the absence of growthsupporting signals at colonization sites. Here, we demonstrated that intravenously administered BMMSCs enhanced the colonization capacity of B16-F10 cells, thereby increasing the incidence of lung metastases. In this study, we adopted a serial intravenous administration of BM-MSCs in order to recapitulate a delivery strategy commonly used for treatment of systemic diseases in clinical trials [22] and also used to enhance cell engraftment in pre-clinical models [23]. After BM-MSCs inoculation in mice previously injected with melanoma cells, the tumor burden in the lungs increased, as well as the number of superficial macrometastases, demonstrating that BM-MSCs enhanced the colonization capacity of B16-F10 melanoma cells. These results are in line with the
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findings of Shinagawa et al., who observed that intrasplenic co-injection of KM12SM colon cancer cells and MSCs leads to the formation of more liver metastases than KM12SM injected alone [24]. On the other hand, Studeny et al. observed that systemic inoculation of human MSCs had no effect on the growth of experimental human breast cancer metastases in the lungs [25]. These discrepancies can be in part attributed to differences on experimental conditions like the use of distinct tumor cell types [12] or the use of immunodeficient mouse models that cannot recapitulate the interaction between MSCs and immune cells, which in turn may have a fundamental role during tumor progression. Of note, the supportive role of BM-MSCs during metastases formation observed in this study is in agreement with previous evidences demonstrating that colonization is not a cell-autonomous process but is rather dependent on the interaction between tumor and stromal cells at colonization sites. For instance, depletion of S100A4+ fibroblasts reduces the metastatic colonization without affecting the growth of primary mammary tumors in mice [26]. In addition, induction of periostin expression in lung stromal cells is required for the establishment of experimental breast cancer metastases in these organs [27]. Although we did not assessed in situ extravasation, it was previously reported that 80% of B16-F10 cells survive and extravasate within 24 h after injection into circulation [28]. Since the first administration of BM-MSCs was given 48 h after B16luc cells inoculation, their effect on colonization is likely to be a consequence of pro-mitogenic/survival stimuli rather than enhancement of extravasation. Accordingly, our in vivo BLI analysis revealed that the number of B16luc cells in the lungs during 8 days following injection was not altered by BM-MSCs administration. In order to shed light on the putative mechanisms underlying the promotion of B16-F10 metastatic outgrowth by BM-MSCs, we investigated whether some potentially pro-tumorigenic properties of BM-MSCs were activated during interaction with B16-F10 cells in vitro or during metastatic colonization in vivo.
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MSCs exert their therapeutic benefits through multiple mechanisms. One of them is through support to the growth of endogenous progenitor cells such as cardiac stem cells [29] and skeletal myoblasts [30]. The mitogenic stimuli provided by MSCs are mediated by secreted molecules such as the vascular endothelial growth factor (VEGF), or by direct cell-to-cell contact interactions [31]. Using direct co-culture assays, we found that BM-MSCs also stimulate the proliferation of B16-F10 melanoma cells in vitro. Moreover, the growth of B16-F10 cells was directly proportional to the number of BM-MSCs seeded in the co-culture assays. Although a clear demonstration of the mitogenic effect of BM-MSCs in vivo is challenging and remains to be done, our observations indicate that BM-MSCs are potentially capable of directly supporting the growth of B16-F10 cells during colonization. Numerous studies reported opposite effects of MSCs on tumor cell proliferation. As examples, human BM-MSCs secrete IL-6 which in turn activate STAT3 in the human osteosarcoma cell line Saos-2 and promote their growth in vitro [32]. Recently, it was demonstrated that MSCs derived from gastric cancer enhance the proliferation of gastric tumor cells through exosomal transference of microRNAs [33]. Conversely, MSCs were reported to suppress the proliferation of K562 leukemia cells through secretion of dickkopf-1 (DKK-1), a negative regulator of the WNT pathway [34]. Therefore, the effect of MSCs on tumor cell proliferation depends on which molecular circuitries are activated or repressed during this heterotypic interaction. As additional properties of their regenerative repertoire, MSCs are known to stimulate angiogenesis and to suppress the activation and proliferation of virtually all cells from immune system, which has broadened their potential clinical application to include the treatment of ischemic and immune-related diseases. However, these MSC properties were also responsible to promote human colorectal tumor growth through induction of angiogenesis [35] and to allow the development of melanoma after transplantation of B16-F10 cells in allogenic recipients through suppression of cytotoxic CD8+ T cells [36]. In the present study, administration of BM-MSCs did not alter the
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number of CD31+ endothelial cells or CD8+ and CD4+ T lymphocytes within metastases-bearing lungs. This indicates that facilitation of B16-F10 metastatic outgrowth was not mediated by induction of angiogenesis or suppression of immunological surveillance. Indeed, the poor immunogenicity of B16-F10 melanoma cells have been long known [37], thus it is expected that immunosuppression of the host is not critical for the metastatic colonization in our model. Tumors have been long conceived as wounds that never heal [38] and they are infiltrated by a myriad of immune cells, stromal cells and endothelial cells that compose the tumor stroma. Infiltrating CD11b+ myeloid cells, which comprise macrophages, neutrophils and monocytes, for example, have been shown to facilitate the acquisition of many hallmarks of cancer, including sustained proliferation, angiogenesis, invasion and metastasis [39, 40]. It was demonstrated that MSCs isolated from spontaneous mouse lymphoma enhances the recruitment of CD11b+ macrophages/monocytes to primary tumors via secretion of CCR2 ligands and that these infiltrating cells accelerate tumor growth [41]. In addition, studies using xenograft or spontaneous models of breast cancer demonstrated that CD11b+ myeloid cells are recruited to specific organs such as the lungs where they form a “premetastatic niche” and facilitate the outgrowth of metastatic tumor cells by promoting vascular remodeling through secretion of metalloproteinases [42] or by stimulating mesenchymal-to-epithelial transition and tumor cell proliferation through deposition of extracellular matrix components such as the proteoglycan versican [43]. In the present study, we demonstrated that systemically administered BM-MSCs enhanced the recruitment of CD11b+ myeloid cells to the lungs with developing melanoma metastases, suggesting an interplay between these cell populations during the promotion of B16-F10 metastatic outgrowth. Infiltrating CD11b+ myeloid cells encompass a broad spectrum of subpopulations whose contributions to the hallmarks of cancer have been extensively uncovered [39]. For instance, CD11b+Gr1+ myeloid cells and tumor-associated macrophages were described to promote tumor neovascularization through release of soluble mediators such as the vascular endothelial growth
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factor (VEGF) [44, 45]. In addition, distinct subsets of CD11b+Gr1+ cells display strong immunosuppressive properties which can favor tumor progression by impairing tumor-specific immune response [46, 47]. Despite these well-described functions, we observed that the higher recruitment of CD11b+ cells to the lungs after MSCs injection did not implicate in changes on the number of endothelial cells and T cell subsets. These correlative evidences indicate that the immunosuppressive and angiogenic functions of CD11b+ myeloid cells were not crucial for their support to metastatic outgrowth in our model. However, alternative mechanistic explanations remain possible such as the release of growth factors after proteolytic cleavage of extracellular matrix by CD11b+ cells-derived proteases [48] and protection against death of tumor cells triggered by integrin signaling during direct heterotypic interaction at colonization sites [49]. Due to their wide repertoire of regenerative properties, MSCs have been investigated to treat an equally broad range of local and systemic diseases such as ischemia, autoimmune diseases and transplant-related complications like the graft-versus-host disease [4]. Of note, MSCs also have a tropism to inflammation sites and tumors [5]. This particular characteristic has driven the use of genetically engineered MSCs to deliver anti-cancer molecules directly to developing tumors [6]. However, our findings demonstrate that systemically administered unmodified BM-MSCs might support the growth of metastases. These are valuable information for the progressive development of BM-MSCs as a safe therapeutic tool and imply that a thorough clinical inspection for the presence of incipient metastases should be carried out in patients before and during the therapy with systemically administered BM-MSCs. Furthermore, if applicable to other tumor types, our observations indicate that the efficiency of the metastatic colonization is affected by the interaction among BM-MSCs, myeloid cells and tumor cells at the target organs.
5. Competing interests The authors have no conflict of interests to declare.
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6. Acknowledgements The authors would like to thank Patrícia V. B. Palma and Camila Menezes for their support with flow citometry analysis. This work was funded with resources from São Paulo Research Foundation (FAPESP – Proc. 2008/08944-0), Coordination for Improvement of Higher Education Personnel (CAPES) and from the National Counsel of Technological and Scientific Development (CNPq Proc. 310619/2012-2).
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8. Figure legends Figure 1: Characterization of bone marrow-derived mesenchymal stromal cells (BM-MSCs). (A) Phase-contrast photomicrographs of BM-MSCs cultures at 3rd and 6th passage. At third passage, BMMSCs cultures were highly heterogeneous, as noticed by the presence of morphologically distinct cell types (left panel). At sixth passage, however, the cultures became morphologically homogeneous and cells displayed multipolar fibroblastic morphology, as expected for BM-MSCs (right panel). Magnification = 100x. (B) Expression of cell surface markers in BM-MSCs cultures evaluated by flow cytometry. At the third passage, BM-MSCs cultures were still contaminated with endothelial (CD31+ cells) and hematopoietic cells (CD11b+, CD45+ and CD34+ cells). However, after six passages in culture, these hematopoietic and endothelial cell contaminants were no longer present. As expected, BM-MSCs did not express the endothelial and hematopoietic markers CD11b, CD31, CD34 and CD45 but were positive for CD44, CD90.2 and Sca-1 (n = 3 independent cultures). (C) Adipogenic differentiation of BM-MSCs. After culture in adipogenic medium for 21 days, BMMSCs accumulated intracellular lipid vesicles, which were stained in orange after staining with Sudan II (left panel). Non-stimulated BM-MSCs did not undergo adipogenic differentiation (right panel). Magnification = 400x. (D) Osteogenic differentiation of BM-MSCs. After culture in osteogenic medium for 21 days, BM-MSCs synthesized mineralized extracellular matrix, which was stained in black or dark brown after staining by von Kossa’s method (left panel,). Cells maintained in regular medium did not undergo osteogenic differentiation (right panel). Magnification = 40x. Figure 2: Systemic inoculation of BM-MSCs facilitates lung colonization by B16luc melanoma cells. (A) Biological activity of luciferase in B16luc melanoma cells. The bioluminescent signal was directly proportional to the number of B16luc cells (linear regression test, R2 = 0.99). (B) Representative images of mice submitted to in vivo bioluminescent imaging to monitor the establishment and growth of melanoma metastases. Systemic administration of BM-MSCs enhanced 20
the lung colonization by B16luc cells (lower pictures) in comparison to mice receiving B16luc cells alone. (C) Quantification of bioluminescent signal from thoracic region of melanoma-bearing mice. Sixteen days after B16luc cells injection, mice treated with BM-MSCs displayed a higher lung metastases burden (blue line) in comparison to mice receiving B16luc cells alone (n = 5 animals per group; *p
