Mesenchymal stem cells generate pericytes to promote tumor recurrence via vasculogenesis after stereotactic body radiation therapy

ARTICLE IN PRESS Cancer Letters ■■ (2016) ■■–■■

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Q2 Original Articles

Mesenchymal stem cells generate pericytes to promote tumor recurrence via vasculogenesis after stereotactic body radiation therapy Q1 Huan-Huan Wang a,1, Yao-Li Cui b,1, Nicholas G. Zaorsky c,1, Jie Lan d, Lei Deng d,

Xian-Liang Zeng a, Zhi-Qiang Wu a, Zhen Tao a, Wen-Hao Guo e, Qing-Xin Wang a, Lu-Jun Zhao a, Zhi-Yong Yuan a, You Lu d, Ping Wang a, Mao-Bin Meng a,* a Department of Radiation Oncology and Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute & Hospital, National Clinical Research Center for Cancer, Tianjin 300060, China b Department of Lymphoma and Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute & Hospital, National Clinical Research Center for Cancer, Tianjin 300060, China c Department of Radiation Oncology, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA d Department of Thoracic Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, West China Clinical Medicine School, Sichuan University, Chengdu, Sichuan 610041, China e Department of Abdominal Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, West China Clinical Medicine School, Sichuan University, Chengdu, Sichuan 610041, China

A R T I C L E

I N F O

Article history: Received 20 November 2015 Received in revised form 30 January 2016 Accepted 17 February 2016 Keywords: Stereotactic body radiation therapy Mesenchymal stem cells Pericytes Vasculogenesis Tumor recurrence

A B S T R A C T

Background: Stereotactic body radiation therapy (SBRT) is postulated to enhance the recruitment of mesenchymal stem cells (MSCs) into the tumor microenvironment, which promote tumor recurrence. The aim of this study is to determine the molecular mechanisms behind SBRT stimulating MSC migration and differentiation. Methods: In vitro, mediated factors and migrated MSCs (post-SBRT) were generated. In vivo, bonemarrow derived MSCs were identified and harvested from green fluorescent protein (GFP)-expressing transgenic male mice and transplanted into sub-lethally irradiated recipient female mice to establish a model of bone marrow transplantation. Lewis lung carcinoma and malignant melanoma-bearing recipient mice were treated with SBRT, 14 Gy/1 fraction. The migration and differentiation potential of MSCs were characterized. Results: SBRT increased the release of stromal cell derived factor-1α (SDF-1α) and platelet-derived growth factor-B (PDGF-B) by tumor cells; these ligands bound to chemokine (C–X–C motif) receptor 4 (CXCR4) and platelet-derived growth factor receptor-β (PDGFR-β), respectively, on circulating bone marrowderived MSCs, resulting in engraftment of the MSCs into the tumor parenchyma. The newly-homed MSCs differentiated into pericytes, which induced the tumor vasculogenesis, and promoted tumor regrowth. Targeted therapies, AMD3100 and imatinib abrogated MSC homing, vasculogenesis, and tumor regrowth. Conclusion: Bone-marrow derived MSCs migrate to the tumor parenchyma and differentiate into pericytes, inducing tumor vasculogenesis after SBRT, and promoting tumor recurrence. MSC migration and maturation may be abrogated with AMD3100 and imatinib. This novel treatment strategy warrants clinical investigation. © 2016 Elsevier Ireland Ltd. All rights reserved.

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Introduction Approximately half of all patients with lung cancer receive radiation therapy (RT) during their disease course [1]. Stereotactic body radiation therapy (SBRT) has allowed clinicians to increase dose de-

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* Corresponding author. Tel.: +86 22 23341405; fax: +86 22 23344105. E-mail address: [email protected] (M.-B. Meng). 1 These authors contributed equally to this work.

livered to the tumor and minimize irradiation of normal tissue, thereby increasing the therapeutic ratio. With SBRT, local and/or regional recurrence is still noted in 10–38% of patients [2–4]. Recurrent tumors are less responsive to salvage treatments (e.g. surgery, RT, systemic therapy) and have a higher risk of metastasis, which impacts patient survival and quality of life [2]. The exploration of the mechanisms behind tumor recurrence after SBRT is essential for the development of novel treatment strategies. The growth, metastases, and therapeutic response of a tumor depend on a functional vascular network [5]. Generally, two

http://dx.doi.org/10.1016/j.canlet.2016.02.033 0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.

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processes contribute to tumor vessel expansion: (1) angiogenesis, the sprouting of new vessels from preexisting vessels; and (2) vasculogenesis, the process by which bone marrow cells are recruited to the tumor and organized to form a vascular network de novo [6]. Vasculogenesis plays a key role in the tumor recurrence after SBRT; inhibiting this pathway may improve local control [7–9]. Vasculogenesis after SBRT depends, in part, on interactions among cells that constitute the blood vessels. Mesenchymal stem cells (MSCs) are multipotent cells, originating from either bone marrow or adipose tissue [10,11], that have the capacity to differentiate into bone, cartilage, muscle, and connective tissues throughout the body [12]. MSCs migrate to sites of tissue (e.g. kidney, heart, and skin), secondary to the local production of inflammatory mediators produced after tissue damage [13,14]. MSCs may contribute to tumor vessel formation to support local recurrence [15]; RT may enhance the recruitment of MSCs into the tumor microenvironment [16–18]. In this study, we explore the relationship between SBRT and MSC migration to the tumor parenchyma. We hypothesize that MSCs differentiate into pericytes, which induce the tumor vasculogenesis, promoting tumor recurrence. We also hypothesize that interfering with recruitment and/or differentiation of MSCs by blocking the stromal cell derived factor 1α (SDF-1α)/chemokine (C–X–C motif) receptor 4 (CXCR4) and platelet-derived growth factor-B (PDGF-B)/ platelet-derived growth factor receptor-β (PDGFR-β) signaling pathways inhibits vasculogenesis and abrogates recurrence after SBRT.

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Cell lines and cell culture

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Lewis lung carcinoma cells (LLCs) and malignant melanoma cells (B16F10), obtained from American Type Culture Collection (ATCC, Rockville, MD, USA), were maintained in 1640 medium (HyClone, Logan, Utah, USA) containing 10% heatinactivated fetal bovine serum (F. Hoffmann-La Roche Ltd, Basel, Switzerland), 100 U/ mL penicillin and 100 μg/mL streptomycin (HyClone, Logan, Utah, USA), and kept in a humidified 5% CO2 atmosphere incubator at 37 °C. Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cord veins using a standard procedure as previously described and grown in EBM-2 medium with SingleQuots™ (Lonza, Walkersville, MD, USA) containing VEGF and other growth factors [19]. HUVECs at passages 5–8 were used for all experiments. The LLCs, B16F10 cells, and HUVECs were irradiated with 14 Gy/1 fraction using a linear accelerator (ELEKTA 1232 Medical Linear Accelerator) at a dose energy of 6 MV-X, with a dose rate of 200 cGy/min, a field size of 24 × 24 cm2, a source to surface distance of 100 cm, and a 2-cm bolus of solid water.

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Animals

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Donor male mice, C57BL/6J-Tg (ACTB-EGFP) 10sb/J, expressing green fluorescent protein (GFP) were offered by the Jackson Laboratory (003291, Bar Harbor, Maine, USA), and the recipient female mice, C57BL/6J, were offered by the State Key Laboratory Biotherapy of Sichuan University (Chengdu, China). All mice were matched for age (8 weeks old) and weight (19.26 ± 1.80 g), and animals were given water and sterile food pellets ad libitum and were kept in laminar air flow benches of the Laboratory Animal Research Center of Tianjin Medical University Cancer Institute & Hospital, National Clinical Research Center for Cancer. All animal care and experimental procedures were approved by and conducted according to Institutional Animal Care and Use Guidelines (No. SYXK 2007-008).

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Isolation and characterization of bone marrow GFP-expressing MSCs

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The MSCs from donor C57BL/6J-Tg male mice were isolated according to previously-reported methods [20]. Briefly, the C57BL/6J-Tg 10sb/J donor male mice were anesthetized by an intramuscular injection of Nembutal at 25 mg/kg. Complete tibia and femurs were extracted, and both ends of the metaphysis were removed. Sterile D-Hanks solution with 100 units/mL heparin was slowly injected into the bone marrow cavity of the tibia, femur, and iliac crest to collect the bone marrow cells (BMCs). After isolation of the NS by the density centrifugation, the Sca1 positive population was separated using magnetic beads (MACS, Miltenyi Biotec, Germany) as recommended by the manufacturer. The specific steps were as follows: 5 × 107 BMCs were centrifuged with 250 g for 10 min and then resuspended carefully in 400 μL sterile MACS buffer. Then, 100 μL Sca1 microbeads for direct separation was added to the sample and incubated for

15 min at 4 °C. After the incubation, cells were washed in 10 mL MACS buffer and again centrifuged at 250 g for 10 min. The cellular pellet was resuspended in 500 μL cooled MACS buffer and loaded onto a separation column in a magnetic field. The flow through cell fraction was preserved and the column was washed 3 times with 500 μL of MACS buffer. The separation column was removed from the magnetic stand and the Sca1 positive cell fraction was eluted with 1 mL MACS buffer. The eluted Sca1 positive cell fraction was tested for cell surface markers by fluorescenceactivated cell sorting (FACS; FACSAria™ Cell Sorter, BD Biosciences) using phycoerythrin (PE)-conjugated antibodies (BD Biosciences). In order to further confirm that the isolated Sca1-positive BMCs were MSCs, differentiation assays were carried out to assess Sca1 positive BMC differentiation into adipogenic, chondrogenic and osteogenic cells.

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Cell migration assay in vitro

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MSC migration assays were performed using the Transwell model in vitro. Briefly, 1 × 105 MSCs as well as AMD3100 (22 mg/mL) or imatinib (20 μM) were placed on Transwell plates with 8 mm pores (BD Biosciences). The LLCs, B16F10, or HUVECs in serum-free media were irradiated with 14 Gy/1 fraction. The media of the irradiated cells, SDF-1α (100 ng/mL), and PDGF-B (500 ng/mL) were transferred to the lower chamber of migration plates after irradiation, and the migration of MSCs was assessed after 24 hours. The migrated cells were then fixed, stained, counted; stained cells were eluted from membranes, and absorbance measurements were performed.

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Whole bone marrow transplantation

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The bone marrow transplantation was performed as previously described [21]. Briefly, after establishment of an optimal sublethal irradiation dose, recipient C57BL/ 6J female mice were sublethally irradiated with 8 Gy/1 fraction 4 hours before reconstitution. Then 1 × 107 donor GFP-expressing MSCs and 3 × 107 recipient whole bone marrow cells suspended in 100 μL PBS and injected via the tail vein of recipient female mice. Control animals received PBS injections. Within 4 weeks, control animals died while transplanted animals survived and displayed donor-derived cells in the bone marrow, which confirmed the successful whole bone marrow transplantation.

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Xenograft mouse model, treatment protocol, and tumor response evaluation

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Four weeks after whole bone marrow transplantation, 2 × 105 LLCs or B16F10 cells were subcutaneously injected into the right rear thigh of recipient mice. Tumor volumes were calculated by the formula π/6 × a × b2, where a is the widest tumor dimension and b is in a plane perpendicular to this dimension. When tumors reached approximately 75–100 mm3, mice were randomly and equally divided among six groups, with eight mice in each group. SBRT, at 14 Gy/1 fraction, was administered at 8 days for xenografts of recipient mice. Then, normal saline, with AMD3100 or imatinib, was injected via the tail vein for two consecutive weeks after SBRT. The regimens were as follows: (i) control (normal saline); (ii) SBRT (14 Gy/1 fraction); (iii) AMD3100 (5 mg/kg); (iv) imatinib (100 mg/kg); (v) SBRT + AMD3100 (14 Gy/1 fraction + 5 mg/kg); (vi) SBRT + imatinib (14 Gy/1 fraction + 100 mg/kg). Before SBRT, mice were anesthetized with chloral hydrate and then restrained on an acrylic board with adhesive tape. With a lead board shielding the mice bodies, only the tumor-bearing legs were irradiated locally at the center of the 24 × 24 cm field. Tumor response was evaluated by a tumor regrowth delay assay; additionally, as an indirect measurement of general toxicity of each treatment regimen, the body weights of mice were monitored. At least five mice from each group were sacrificed by cervical dislocation on the planned days after SBRT, and further analyses focusing on changes in the tumor vasculogenesis were performed.

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Materials and methods

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Detection of male MSCs in female xenografts after SBRT

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The tumor tissues from the recipient female mice were subjected to DNA extraction (Omega) and real-time PCR analysis to detect the presence of male MSCs. The samples (200 ng) were examined for real-time polymerase chain reaction (realtime PCR) with an SYBR PCR kit. Primer sequences are provided in supplementary material. Male mouse DNA was identified by the presence of Y6, while total mouse DNA was identified by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Standard curves of Y6 and GAPDH genes generated by diluting male genomic DNA into female genomic DNA (at a ratio from 1:10,000 to 400:1 and from 10:1 to 500:1, respectively) were used as a reference for unknown DNA samples. The DNA and copy number were proportional to the number of cells. Each cell (diploid) contained 6.16 pg DNA for two copies of non-repeated gene (GAPDH), and 3.08 pg DNA for one copy of non-repeated gene (Y6). The results were expressed as the number of male cells per 100 female cells.

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Enzyme-linked immunosorbent assay (ELISA)

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The tumor cells and endothelial cells with condition supernatants were harvested at 0, 1, 6, 12, 24, 48, and 72 hours after SBRT in vitro; serum was collected in non-anticoagulation unfertile tubes from mice at 0, 1, 6, 12, 24, 48, 72 and 120 hours after SBRT in vivo. The blood samples were centrifuged at 3000 r/min for 5 minutes at 4 °C, and the separated serum samples were stored at −20 °C until use

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for assay. The SDF-1α and PDGF-B levels were measured using the monoclonal antimouse capture antibody (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Calibration curves were established with known concentration of the angiogenic factors, each microtiter plate with a standard curve. The colorimetric reaction was read with a Benchmark Microplate Reader (Benchmark Electronics, Inc., Angleton, TX, USA).

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Western blotting (WB)

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Protein was extracted from cells and tumor tissues. Equivalent amounts of protein were separated by 15% SDS–PAGE gel and transferred to a PVDF membrane (Millipore). Membranes were incubated with rabbit anti-mouse polyclonal SDF-1α, CXCR4, PDGFB, and PDGFR-β. Then, membranes were incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Antibody binding was detected using the electrochemiluminescence (ECL) detection kit to produce a chemiluminescence signal, which was captured on x-ray film. Relative protein expression levels were calculated relative to β-actin.

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Immunofluorescence (IF) staining

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To confirm α-SMA, PDGFR-β, and neuro/glial cell 2 chondroitin sulfate proteoglycan (NG2) staining cells represented perivascular pericytes, IF was used to co-localize the pericyte markers staining with platelet/endothelial cell adhesion molecule-1 (PECAM-1, endothelial cell marker) staining. IF staining was briefly conducted on frozen section of tumor tissues with visual combination of dual staining of rabbit anti-mouse monoclonal PECAM-1 antibody (1:200) and rabbit antimouse polyclonal α-SMA, PDGFR-β and NG2 antibody (1:100). The secondary antibody detection was performed using FITC-conjugated goat anti-rabbit for PECAM-1 (1:100) and contrasting TRITC-conjugated goat anti-rabbit for α-SMA, PDGFR-β, and NG2 (1:100). For quantification of IF staining, five slides per tumor with 5 randomly selected regions per slide and 5 tumors of each group were observed. The microvessel density (MVD) of the tumor was measured by scanning the stained section of PECAM-1 under high-power field (200-fold magnification). The ratio of α-SMA/ PECAM-1, NG2/PECAM-1, and PDGFR-β/PECAM-1 was calculated by dividing the positive area of α-SMA, NG2, and PDGFR-β adjacent to PECAM-1 positive vessels by the total area of PECAM-1 positive tumor vasculature under five high powered (200fold magnification) randomly chosen fields per slide. In addition, the specific receptors CXCR4 and PDGFR-β of SDF-1α and PDGF-B were also evaluated by IF staining in MSCs. The specimens were examined with an inverted fluorescence microscope (IX50, Olympus, Japan).

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Data analysis

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Continuous variables were expressed as the mean ± standard deviation (SD), and these were compared using an unpaired Student’s T-test. Tumor regrowth was compared using repeated measures one-way analysis of variance (R M-ANOVA). The differences with p < 0.05 (two-tailed) were considered statistically significant. Data were analyzed using the statistical software Intercooled Stata version 8.2 for Windows (Stata Corporation, College Station, Texas, USA).

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Results The identification of bone marrow MSCs and whole bone marrow transplantation The isolated bone marrow GFP-expressing MSCs exhibited a spindle-shaped morphology in culture; MSCs could be induced to differentiate into osteocytes, adipocytes, and chondrocytes (Fig. 1A). Similar to typical MSCs, fluorescence activated cell sorting (FACS) analysis revealed that the MSCs derived were characterized by expression of CD44 and CD29 and non-expression of CD31, CD45, and CD11b (data not shown). These results suggest that the isolated bone marrow GFP-expressing BMCs consisted of MSCs in vivo. To address whether the MSCs homed into the tumor environment after SBRT in vivo, whole bone marrow transplantation was performed to track green fluorescent protein (GFP)-expressing MSCs and/or its progeny cells (Fig. 1B). The recipient mice did not have GFP-expressing cells in the bone marrow before bone marrow transplantation; however, 41.45% ± 0.87% of GFP-expressing MSCs and/ or its progeny cells were present in the bone marrow after stable reconstitution bone marrow of recipient female mice, suggesting that bone marrow transplantation was successful (Fig. 1C).

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SBRT enhances the migration of MSCs by SDF-1α and PDGF-B in vitro In vitro, the migration assay was performed to determine the radiation-induced tumor cell and/or endothelial cell factors responsible for MSC migration. Our results revealed that the expression of SDF-1α and PDGF-B in the tumor-condition supernatants significantly increased compared to HUVECs-condition medium at 6 hours after SBRT. The expression of these two factors in the tumorcondition supernatants reached a peak at 24 hours; however, no significant difference was observed between these two cell lines (Fig. 2A–C). Additionally, MSCs expressed specific receptors, CXCR4 and PDGFR-β, for secreted proteins SDF-1α and PDGF-B, respectively (Fig. 2D and E). We assessed the impact of the SDF-1α/CXCR4 and PDGF-B/ PDGFR-β interaction on the migration capacity of MSCs with tumorcondition supernatants and/or HUVECs-condition supernatants in vitro (Fig. 2F). The results showed that under the induction of the tumor-condition supernatants as well as agonists including SDF1α and PDGF-B after SBRT, the number of MSCs passing through the microporous membrane was significantly higher than that of the HUVECs-condition supernatants (p < 0.05). When the specific inhibitors (AMD3100 and imatinib; which inhibit CXCR4 and PDGFR-β on MSCs, respectively) were added, the number of MSCs passing through the microporous membrane decreased significantly by 17.5%–45.5%, compared to that of the tumor-condition supernatants and agonists (all p < 0.05, Fig. 2G–M). The findings suggest that SBRT induced and enhanced the migration potential of MSCs by SDF-1α and PDGF-B in vitro; AMD3100 and imatinib abrogated the migration. To assess the role of angiogenesis in tumor recurrence, we performed additional experiments using an ELISA on serum from xenograft models after SBRT to determine VEGF expression levels. Our results revealed that SBRT decreased the release of VEGF in a time-dependent manner (Fig. S1).

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SBRT enhances the recruitment of MSCs into the tumor parenchyma in vivo The rate of donor MSC migration in the tumor and other tissues of recipient mice was quantified within 120 hours after SBRT. Standard curves of Y6 (representing donor MSCs DNA) and GAPDH (representing total mouse DNA), generated by diluting male genomic DNA into recipient genomic DNA, were used as reference controls (Fig. 3A and B). The recruitment of MSCs into the tumor microenvironment after SBRT was time-dependent from 0.028 ± 0.009% at 6 hours to 0.132 ± 0.019% at 120 hours (Fig. 3C). Meanwhile, a higher percentage of GFP-expressing MSCs and/or its progeny cells was detected in the tumor microenvironment of the recipient mice as compared to in the heart (0.132% versus 0.004%), and lower in other tissues (Fig. 3D). We assessed the impact of the SDF-1α/CXCR4 and PDGF-B/ PDGFR-β interaction on the recruitment of MSCs into tumor microenvironment in vivo. Among both LLC and B16F10 xenografts, SBRT significantly increased SDF-1α and PDGF-B expression in serum compared to tumor tissues, and the expression reached a peak at 24 hours after SBRT initiation (Fig. 3E–H). Furthermore, SBRT significantly increased the recruitment of MSCs into the tumor parenchyma in LLCs and B16F10 xenografts. Similar results were observed in vitro after the specific inhibitors (AMD3100 and imatinib) were added: the recruitment of MSCs decreased significantly compared to that of SBRT alone (Fig. 3I). These data suggested that SBRT enhanced the recruitment of MSCs into the tumor parenchyma by the SDF-1α/CXCR4 and PDGF-B/PDGFR-β signaling pathways in vivo.

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Fig. 1. Characterization of MSCs derived from bone marrow of mice with stain C57BL/6J-Tg and whole bone marrow transplant experimental designs. (A) The isolated bone marrow Scal+-MSCs exhibited a spindle-shaped morphology in culture. The MSCs could be induced to differentiate into osteocytes, adipocytes, and chondrocytes. (B) The GFP+-expressing MSCs from male mice (C57BL/6J-Tg) plus whole marrow cells from female receipt mice (C57BL/6) were transplanted into sublethally irradiated female receipt mice. After 4 weeks, the female bone marrow was obtained, and engraftment was verified by 30% GFP expression in bone marrow, which confirmed the successful whole bone marrow transplantation. On the planned days, the xenografts were resected and further analyses were performed, focusing on GFP-expressing MSC migration, differentiation, and vasculogenesis. (C) The recipient mice did not have GFP-expressing cells in the bone marrow before bone marrow transplantation; however, 41.45% ± 0.87% of GFP-expressing MSCs and/or its progeny cells were present in the bone marrow after stable reconstitution bone marrow of recipient female mice. MSCs: mesenchymal stem cells; GFP: green fluorescent protein.

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SBRT decreases microvascular density but enhances pericyte coverage of the endothelial cells The tumor vascular dynamic changes after SBRT were investigated by staining the endothelial cells and the pericytes in LLC xenografts. The MVD of xenografts in SBRT group was significantly reduced compared to that in the control group, 59.7 vs. 34.7 for LLCs, and 51.22 vs. 37.8 for B16F10 cells (p = 0.0001 and p = 0.01, Fig. 4A and B). There was scarce pericyte coverage on endothelial cells, and pericytes were loosely associated with the endothelial cells in the control group; however, the surviving vessels in SBRT tumors were more dilated, less branching, and closely associated with α-SMA, NG2, and PDGFR-β positive pericytes. For example, the ratio of α-SMA/PECAM-1 increased significantly from 0.15 to 0.24 after SBRT (p = 0.03); however, with addition of the specific agonists, AMD 3100 or imatinib, the ratios were significantly lower (at 0.17 and 0.20, respectively) vs. SBRT alone (p = 0.02 and p = 0.07). The ratio of NG2/PECAM-1 increased significantly from 0.17 to 0.38 after SBRT (p = 0.0003); however, with addition of the specific agonists, AMD 3100 or imatinib, the ratios were significantly lower (at 0.25 and

0.29, respectively) vs. SBRT alone (p = 0.02 and p = 0.04). The ratio of PDGFR-β/PECAM-1 increased significantly from 0.16 to 0.42 after SBRT (p = 0.00001); however, with addition of the specific agonists, AMD 3100 or imatinib, the ratios were significantly lower (at 0.21 and 0.32, respectively) vs. SBRT alone (p = 0.0003 and p = 0.01). The data suggest that SBRT caused a decrease in MVD and contributed to increasing pericyte coverage of the vessel endothelium via the SDF-1α/CXCR4 and PDGF-B/PDGFR-β signaling pathways. MSCs differentiate into pericytes, which induced vasculogenesis and promoted tumor recurrence Consistently, SBRT enhanced recruitment of MSCs into the tumor parenchyma; additionally, SBRT enhanced pericyte coverage of the endothelial cells by the SDF-1α/CXCR4 and PDGF-B/PDGFR-β signaling pathways. Therefore, we speculated that there may be interaction between the pericytes and MSCs after SBRT. Our results showed that the pericyte marker NG2 partially co-localized with GFP-expressing MSCs, while PECAM-1 (a representative endothelial marker) did not localize, but was in close contact with

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Fig. 2. SBRT enhances the migration of MSCs by SDF-1α and PDGF-B in vitro. (A) The expression of SDF-1α and PDGF-B in tumor cells and endothelial cells was evaluated after SBRT at planning time by Western blotting. (B, C) The expressions of SDF-1α and PDGF-B in the tumor and endothelial cell condition supernatants were evaluated after SBRT by ELISA. (D, E) The expression of the specific receptors CXCR4 and PDGFR-β for SDF-1α and PDGF-B was evaluated by Western blotting and IF. (F) MSC migration assay was performed using the Transwell assay in vitro. Briefly, the MSCs as well as AMD3100 or imatinib were placed on Transwell plates, the media of the irradiated tumor cells and endothelial cells, SDF-1α, or PDGF-B as transferred to the lower chamber of migration. The migration of MSCs was assessed after 24 hours. (G–M) The impact of the SDF-1α/CXCR4 and PDGF-B/PDGFR-β interaction on the migration of MSCs in the tumor and endothelial cell condition supernatants was evaluated. SBRT: stereotactic body radiation therapy; SDF-1α: stromal cell derived factor 1α; PDGF-B: platelet-derived growth factor-B; ELISA: enzyme-linked immunosorbent assay; CXCR4: chemokine (C–X–C motif) receptor 4; PDGFR-β: platelet-derived growth factor receptor-β; IF: immunofluorescence staining.

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Fig. 3. SBRT enhances the recruitment of MSCs into the tumor parenchyma in vivo. (A, B) Standard curves of Y6 (representing donor MSCs DNA) and GAPDH (representing total mouse DNA), generated by diluting male genomic DNA into recipient genomic DNA, were used as reference controls. (C, D) The rates of donor MSCs in the tumor and other tissues of recipient mice within 120 hours were quantified after SBRT by real-time PCR. (E, F) The expression of SDF-1α and PDGF-B in xenografts was evaluated after SBRT at planned time by Western blotting. (G, H) The expression of SDF-1α and PDGF-B in serum was evaluated by ELISA. (I) The impact of the SDF-1α/CXCR4 and PDGFB/PDGFR-β interaction on the recruitment of MSCs into tumor microenvironment was evaluated. PCR: polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

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GFP-expressing cells at the perivascular niche (Fig. 5A). These observations suggest that the homing of bone-marrow derived MSCs differentiated into pericytes. To determine whether selective elimination of MSC–pericyte interaction on xenograft regrowth delay could be achieved, we investigated combination therapy with SBRT and the specific inhibitors AMD3100 and imatinib. The combination of SBRT + AMD3100 as well as SBRT + imatinib led to significantly delayed tumor growth compared with SBRT alone (all p < 0.05); furthermore, SBRT + AMD3100 led to significantly delayed tumor regrowth compared to the SBRT + imatinib combination schedule (p < 0.05, Fig. 5B and C). After plotting log-transformed tumorgrowth curves to assess tumor-growth rates, the tumor-growth rate curves of LLCs and B16F10 xenografts grown in each group diverged, especially in B16F10 xenograft (Fig. 5D and E). The results suggest that SBRT induced the release of SDF-1α and PDGF-B. The binding of SDF-1α to CXCR4 and PDGF-B to PDGFR-β induced the migration of bone marrow-derived MSCs into the tumor parenchyma and differentiation into pericytes after SBRT, and promoting tumor recurrence. MSC migration and maturation may be

abrogated with AMD3100 and imatinib. An illustration of the mechanism is provided (Fig. 6). Discussion As tumors expand in size, the tumor and local parenchyma become incapable of supplying blood to the tissue. Investigators have studied whether tumor endothelium is formed by angiogenesis, remodeling by surrounding tissue vessels, or vasculogenesis (i.e. the recruitment of bone marrow-derived MSCs into tumors and differentiation of these cells in the tumor microenvironment) [22,23]. Furthermore, studies implicated bone marrow-derived MSCs in vasculogenesis. MSCs have been cultured and injected back into mice to show that they possess the capacity to promote tumor growth [11,24,25]. However, little is known about the role and molecular mechanisms of MSCs in local and/or regional recurrence. The findings in the present study suggest that SBRT increases the release of SDF-1α and PDGF-B by tumor cells; these ligands bind to CXCR4 and PDGFR-β (respectively) on circulating bone marrowderived MSCs, resulting in engraftment of the MSCs into the tumor

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Fig. 4. SBRT decreases MVD but enhances pericyte coverage of the endothelial cells by the SDF-1α/CXCR4 and PDGF-B/PDGFR-β signaling pathways. (A, B) The MVD of xenografts in SBRT group was significantly reduced compared to that in the control group. (C, D) There was scarce pericyte coverage on endothelial cells, and pericytes were loosely associated with the endothelial cells in the control group in LLC xenografts. Importantly, the surviving vessels in SBRT tumors were more dilated, less branching, and closely associated with α-SMA, NG2, and Desmin positive pericytes. When the specific inhibitors, AMD3100 and imatinib, were added into SBRT group, there was a persistent decline in pericyte coverage on endothelial cells compared to SBRT group in LLC xenografts.

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parenchyma. The newly-homed MSCs differentiate into pericytes, which induce the tumor vasculogenesis, and promote tumor recurrence. Targeted therapies, AMD3100 and imatinib, which inhibit CXCR4 and PDGFR-β on MSCs, abrogate MSC homing, vasculogenesis, and tumor recurrence. These data provide a new potential strategy to enhance personalized therapy of SBRT. MSCs are multipotent bone marrow-derived stem cells that migrate to sites of tissue injury, including the kidney, heart, and skin, primarily as a result of the local production of inflammatory mediators from tissue damage and remodeling [13,14]. MSCs have the capacity to differentiate into bone, cartilage, muscle, and connective tissues throughout the body [26,27]. The ability of MSCs to differentiate into many tissues, combined with the facts that MSCs are relatively easy to isolate and are genetically stabile when expanded in culture (Fig. 1), has promoted great interest in using MSCs for tumor vasculature studies. In addition, MSCs are an important component of the tumor microenvironment; however, previous studies have produced controversial results regarding whether MSCs promote or inhibit tumor growth and progression [28–30]. Therefore, we postulated that MSCs may home to the irradiated tumor and differentiate into pericytes; we explored the mechanism behind this process. After SBRT, tumor cells increased the production and secretion of cytokines including SDF-1α and PDGF-B (Fig. 2). The ligands have been shown to play a key role in mediating MSC chemotaxis in vitro. Additionally, studies have shown that a number of inflammatory cytokines (including CXCR1, CXCR2, CCR2, TNF-α and MMP-2) stimulate MSC migration [31–34]; these cytokines are also up-regulated by RT [35,36]. Combined with our results on angiogenesis in this study (Fig. S1) and our previously published results [37,38], we pos-

tulate that the decreasing VEGF levels after SBRT may be due to 488 repression of MVD. Taken together, these results suggest that 489 490 vasculogenesis, and not angiogenesis, plays a key role in tumor re491 currence after SBRT. Inhibiting the vasculogenesis pathway after 492 tumor irradiation has the potential to improve local control. 493 To learn whether MSCs could specifically home to the tumor mi494 croenvironment after SBRT in vivo, whole bone marrow 495 transplantation was performed to track GFP-expressing MSCs (Fig. 1). Our results showed that the recruitment of MSCs into the tumor 496 parenchyma after SBRT was time-dependent, with the majority of 497 homing occurring within 120 hours, decreasing over time (Fig. 3C). 498 Some studies demonstrated that MSCs may be detected in tumor 499 after irradiated at doses of 2 Gy, but there was no difference in the 500 absolute levels of MSC engraftment at day 6 or later [17,39]. Based Q7 501 on the above, our subsequent experiments in vivo were per502 formed after 120 hours of SBRT. Meanwhile, a higher percentage 503 of GFP-expressing MSCs and/or its progeny cells was detected in the 504 tumor microenvironment of the recipient mice as compared to in 505 the heart (0.132% versus 0.004%), and lower in other tissues in506 cluding liver (0.001%), kidney (0.002%), spleen (0.0003%), and brain 507 (0.002%) (Fig. 3D). 508 Likewise, consistent results in vitro, increases in the cytokines 509 SDF-1α and PDGF-B were up-regulated in the irradiated serum, and 510 their expression reached a peak at 24 hours after SBRT (Fig. 3G–H). 511 SBRT directly increased engraftment of bone-marrow derived MSC 512 into the tumor parenchyma. When the specific inhibitors AMD3100 513 and imatinib were added, the homing of MSCs decreased signifi514 cantly (Fig. 3I). Importantly, our findings showed that the tumor MVD 515 decreased and the number of pericyte-covered microvessels in516 creased after SBRT via the SDF-1α/CXCR4 and PDGF-B/PDGFR-β 517

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518 Q12 Fig. 5. The bone-marrow derived MSCs migrated to the tumor parenchyma and differentiated into pericytes, which promoted tumor regrowth and radioresistance. (A) Immunofluorescence staining analysis of pericyte marker NG2 expression in differentiated cells from Sal+-GFP expressing MSCs after SBRT (arrow). (B, C) Tumor regrowth in 519 520 mice bearing LLCs and B16F10 cell xenografts treated with SBRT and/or the specific inhibitors, AMD3100 and imatinib. (D, E) After plotting log-transformed tumor-growth 521 curves to assess tumor-growth rates, it became evident that the tumor-growth rate curves of LLCs and B16F10 xenografts grown in each group diverged, especially in B16F10 522 xenograft. NG2: neuro/glial cell 2 chondroitin sulfate proteoglycan. 523 524 signaling pathways (Fig. 4A–D). These findings concur with our pub525 lished data, which suggest that SBRT induces normalization of tumor 526 vasculature [37,38,40,41]. Recent studies have highlighted the prevalence of the differen527 tiation of bone-marrow cells into periendothelial vascular mural and 528 hematopoietic effector cells de novo during wound healing and tumor 529 530 Q8 vasculogenesis [42–44]. Crisan et al. documented a subpopulation of human perivascular cells with markers common to both pericytes 531 and MSCs in situ [45,46]. These results revealed that blood vessel 532 walls harbor a reserve of progenitor cells that may be integral to 533 the origin of the MSCs and other related adult stem cells; however, 534 the discordant findings likely reflect different experimental tumors 535 used, which have variable ability in their recruitment of different 536 progenitor cells into tumor vessels. 537 Pericytes envelope the vascular endothelium throughout the 538 body; pericytes are important in vessel integrity, stabilization and 539 maturation. Vascular normalization, or the restoration of normal 540 structure and function in blood vessels, is an emerging cancer treat541

ment strategy [47]. Therefore, the tumor vascular dynamic changes (i.e. the MVD) were investigated after SBRT by staining the endothelial cells and pericytes (Fig. 5). The results demonstrated that SBRT decreases MVD; additionally, SBRT contributed to increased pericyte coverage of the vessel endothelium, consistent with our previously-published data [17,39]. Factors regulating tumor recruitment and transformation of bone marrow-derived MSCs into pericytes must also be identified because the pericytes are essential for microvascular stability and recurrence of tumors. Our study demonstrates that SBRT enhanced pericyte coverage of the endothelial cells via the SDF-1α/CXCR4 and PDGF-B/PDGFR-β signaling pathways (Fig. 4A–D). Handan et al. dem- Q9 onstrated that SDF-1α in the tumor microenvironment plays a critical role in promoting pericyte formation in Ewing sarcoma tumor neovascularization by regulating PDGF-B expression. Interfering with this pathway affects tumor vascular morphology and expansion [48,49]. Further studies are warranted to determine the intrinsic molecular mechanisms of regulation of pericyte formation.

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Fig. 6. Diagrammatic illustrations of signaling pathways involved. SBRT increases release of SDF-1α and PDGF-B by tumor cells; these ligands bind to CXCR4 and PDGFR-β (respectively) on circulating bone marrow-derived MSCs, resulting in engraftment of the MSCs into the tumor parenchyma. The newly-homed MSCs differentiate into pericytes, which induce the tumor vasculogenesis, and promote tumor recurrence. Targeted therapies, AMD3100 and imatinib, which inhibit CXCR4 and PDGFR-β on MSCs, abrogate MSC homing, vasculogenesis, and tumor regrowth.

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Vasculogenesis is a process of blood vessel formation that occurs secondary to the migration of progenitor cells in response to a stimulus, and differentiation of those progenitor cells into mature pericytes. During vasculogenesis, pericytes are recruited to surround the endothelial cells and stimulate them to proliferate. Pericytes fulfill at least two important roles in assisting the vasculogenesis: synthesis of growth factors, which may affect other cells by paracrine signaling [50,51]; and formation of the mural wall of new blood vessels [52,53]; neither of these mechanisms are wellunderstood. A number of studies have proved that direct contact and communication between endothelial cells and pericytes/ smooth muscle cells are essential to vascularization [53,54]. We were interested in studying these interactions. Consistently, our results demonstrated that SBRT enhanced the recruitment of MSCs into the tumor parenchyma (Fig. 3); additionally, SBRT enhanced pericyte coverage of the endothelial cells by the SDF-1α/CXCR4 and PDGF-B/PDGFR-β signaling pathways in vivo (Fig. 4). Crisan et al. [55] reported a subpopulation of human perivascular cells that express both pericyte and MSC markers in situ. The isolated population can expand and is clonally multipotent in culture, suggesting that MSCs found from young age to adulthood are of the same pericyte family of cells. However, Caplan [46] believed that pericytes were not MSCs, since both large and small vessels were surrounded by perivascular cells with highly differentiated functions, which are distinct from the activities associated with the osteo-, chondro-, or adipogenic progeny of most MSCs. Furthermore, some investigators found that tumor cell-induced MSCs differentiated into pericytes that enhance neovascularization, and inhibit this process, preventing tumor growth and metastasis and increasing efficacy of anti-cancer therapies [56–58]. Others also found that different stem cells generate vascular pericytes to support vessel function and tumor growth [59,60]. These findings encouraged us to believe that the interaction between the pericytes and MSCs came from bone marrow-derived MSCs after SBRT. The bone-marrow derived MSCs homed to the tumor, and the MSCs differentiated into pericytes, inducing vasculogenesis. Selectively inhibiting the homing or maturation process inhibited tumor recurrence (Figs. 5 and 6). There are some limitations in this study. First, only two cell lines were used in this experiment: LLCs and B16F10 cells. These were chosen because of their high cell proliferation, high blood supply, and resistance to various therapies. Our results should be confirmed in other cell lines. Secondly, a single large-dose SBRT (i.e.

14 Gy) was employed to exclude the potential confounding effects of the conventionally fractionated RT (i.e. 1.8 Gy). Additional studies are needed to explore the intrinsic molecular mechanisms of differentiation of MSCs to pericytes after conventional fractionated RT and SBRT. Next, only 0.132% of MSCs were detected in the tumor microenvironment, which was derived from transplantation MSCs, suggesting that another origin and other factors were involved [11,61]; thus, further studies are needed before therapeutic targeting of bone marrow-derived MSCs in individualized cancer therapy. Additionally, DNA DSB repair proteins of cancer cells may be responsible for SBRT resistance. In summary, SBRT increases the release of SDF-1α and PDGF-B by tumor cells; these ligands bind to CXCR4 and PDGFR-β (respectively) on circulating bone marrow-derived MSCs, resulting in engraftment of the MSCs into the tumor parenchyma. The newlyhomed MSCs differentiate into pericytes, which induce the tumor vasculogenesis, and promote tumor recurrence. Targeted therapies, AMD3100 and imatinib, which inhibit CXCR4 and PDGFR-β on MSCs, abrogate MSC homing, vasculogenesis, and tumor regrowth. These data provide a new potential strategy to enhance personalized therapy of SBRT. Funding This work was supported by the National Natural Science Foundation of China (No. 81201754), the New Teacher Fund for Doctor Station from the Ministry of Education of the People’s Republic of China (No. 20121202120014), and the Foundation of Tianjin Public Health Bureau (No. 2012KZ067). No benefits in any form have been or will be received from a commercial party directly or indirectly related to the subject of this article. Approval/Disclosures All authors have read and approved the manuscript. We have no financial disclosures. We are not using any copyrighted information in this paper. No text, text boxes, or figures in this article have been previously published or owned by another party. Acknowledgements We are indebted to all colleagues at the Laboratory of Cancer Cell Biology of Tianjin Medical University Cancer Institute & Hospital who

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Please cite this article in press as: Huan-Huan Wang, et al., Mesenchymal stem cells generate pericytes to promote tumor recurrence via vasculogenesis after stereotactic body radiation therapy, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.02.033

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